FT 

MEADE 

QP 801 
.A25 H8 
1921 
Copy 2 





4 






* 




4 


✓ 






















/ 









■ 





















































































, 

J 











THE DETERMINATION OF THE ACETONE 
BODIES IN BLOOD, URINE AND EX¬ 
PIRED AIR 


THE EFFECT OF DIETS HIGH IN FAT UPON 
THE EXCRETION OF THE ACETONE 
BODIES 


BY 

ROGER S. HUBBARD 

1 / 






























QHo\ 














































• I^Z5r I % 

\^U 
C-o py 2. 


6/fJ . 

umm< ry 

: '*r 29 










0 

*) *> «> 
*> «> 
o t) «> 

1 « c > 

























* • 






















/ 


.*>• 







The publications included here formed the material presented 
as a thesis in partial fulfilment of the requirements for the 
degree of Doctor of Philosophy in Biological Chemistry at 
Washington University, St. Louis, Missouri in June 1921. 
The work upon the determinations in blood and urine was car¬ 
ried out in the laboratory of biological chemistry at the Wash¬ 
ington University Medical School under the immediate super¬ 
vision of Dr. Philip A. Shaffer, and the larger part of the 
metabolic work was done in the Clifton Springs Sanitarium, 
Clifton Springs, New York. My thanks are due to Dr. Shaf¬ 
fer for his advice and assistance throughout the work, to Dr. 
Floyd R, Wright and Dr. Samuel T. Nicholson jr., of Clifton 
Springs for aid in obtaining suitable material for the metabolic 
experiments reported, and to Dr. Malcolm S. Woodbury, late 
superintendent of the Clifton Springs Sanitarium, for the en¬ 
couragement which he extended while the work was being car¬ 
ried out in that institution. I wish also to express my apprecia¬ 
tion of the necessary assistance given by other physicians, by 
nurses, and by dietitians both in St. Louis and in Clifton 
Springs. 



Reprinted from The Journal of Biological Chemistry, Vol. XLIII, No. 1, 1920 


DETERMINATION OF MINUTE AMOUNTS OF ACETONE 
BY TITRATION.* 

By ROGER S. HUBBARD. 

{From the Laboratory of the Department of Biological Chemistry, Washington 
University, St. Louis, and the Laboratory of the Clifton Springs 
Sanitarium, Clifton Springs.) 

(Received for publication, June 1, 1920.) 

In 1916 a study of the occurrence of acetone bodies in normal 
blood and urine was undertaken. As a preliminary investigation, 
a comparison of the different methods of the determination of 
minute amounts of acetone was made, and certain advantages 
which a volumetric method possesses over a nephelometric 
(Marriott, 1913-14 h) or a gravimetric one 1 (Van Slyke, 1917) led 
to the study and extension of the Messinger method given below. 
Since this modification was first described, various papers have 
appeared (Ljungdahl (1917), Lenk (1916), Richter-Quittner 
(1919)) dealing with the same subject. 

Many different methods have been proposed for the deter¬ 
mination of acetone, and some of these have been adapted to 
minute quantities of acetone. The method of Scott-Wilson (1911) 
depends on the formation of a double compound of acetone and 
mercuric cyanide (Marsh and Fleming-Struthers, 1905), and sub¬ 
sequent titration of the mercury present in the precipitate formed 
by a modified Volhard technique. This method has been used 
for the determination of very small amounts of acetone by Mar¬ 
riott (1913-14 6), who measured the precipitate with a nephelo- 
meter, and by Folin and Denis (1914), who use the colorimeter 
for the same purpose. In 1898 Deniges proposed a method 
depending on the formation of a compound of acetone and mer- 

* A preliminary report of this method was made before the American 
Society of Biological Chemists in New York in 1916 (Hubbard, 1917). 

1 When this paper was reported, only a preliminary report of Dr. Van 
Slyke’s method was available. 


43 


44 


Determination of Acetone 


curie sulfate. The compound was crystalline, and could be 
weighed, or could be decomposed and the mercury determined 
by titrating with silver nitrate and potassium cyanide. This 
method has been studied by Oppenheimer (1899) and by Sammett 
(1913) and has recently been adapted by Van Slyke (1917) for 
the determination of minute quantities of acetone, who recom¬ 
mended either gravimetric determination of the final product, 
or the titration of the mercury by the method of Personne (1863). 
Engfeldt (1915) proposed a colorimetric method for this deter¬ 
mination based on the qualitative method of Frommer (1905), 
using a color reaction given by acetone and salicylic aldehyde. 
Csonka (1916) studied this method, but did not find it applicable 
to very small amounts of acetone. Other reactions are available 
for demonstrating the presence of small amounts of acetone, and 
for determining large amounts quantitatively, but are not well 
adapted for the quantitative determination of very small amounts. 

The Messinger (1888) titration method has been most used 
for the determination of acetone in biological work. This method 
depends on the formation of iodoform from acetone in an alkaline 
iodine solution. A known amount of standard iodine solution 
reacts with acetone in alkaline solution, and the excess of iodine 
is determined, after acidifying, with a standardized thiosulfate 
solution. The method based on an older one of Kramer (1880), 
in which the iodoform was weighed, was proposed by Messinger 
in 1888, and has since been much studied. The principal objec¬ 
tion to it is the number of compounds which give a similar reaction. 

Collischonn (1890) believed that the method was inaccurate 
for small quantities of acetone, but Geelmuyden (1896), Marriott 
(1913-14 a), and others came to the opposite conclusion. Marriott 
(1913-14 a) weighed out small amounts of pure acetone, diluted 
them with water, and found that values obtained on titration 
agreed with the theoretical ones. This work was done with 
solutions of iodine and thiosulfate of approximately 0.1 n con¬ 
centration. 

A few papers have appeared recently in which the use of more 
dilute solutions is described. Lenk (1916), Ljungdahl (1917), and 
Richter-Quittner (1919) have mentioned the use of such solu¬ 
tions, but have not described precautions necessary if they are 
to be used successfully. In a more recent paper Ljungdahl 


R. S. Hubbard 


45 


(1919) has described some of these precautions. He recommends 
the use of the potassium biiodate solution described by Bang 
(1913) for the determination of blood sugar, and gives in detail 
many of the conditions which must be complied with if its use is 
to be satisfactory. Some of these conditions are the same as 
those found in working with iodine solutions prepared by the 
technique described in this paper. A comparison of solutions of 
biiodate with iodine solutions was made in the course of this 
work, and led to the conclusion that fewer precautions were 
necessary when iodine solutions prepared as described were 
used than when biiodate solutions were used. 

0.01 n solutions of iodine cannot be accurately titrated with 
thiosulfate without the addition of an excess of potassium iodide 
(Treadwell, 1915), and 0.001 n solutions are even more unsatis¬ 
factory. It was found that if a solution of iodine in 3 per cent 
potassium iodide was used results of titrations were accurate. 
These solutions were prepared as follows: 

A stock solution of iodine in potassium iodide was made by dissolving 
13.13 gm. of iodine and 25 gm. of potassium iodide in 1 liter of water, and a 
solution of sodium thiosulfate of a corresponding strength by dissolving 
25.65 gm. of the reagent in 1 liter of water. The solutions are 0.1 n X 103.47 
per cent and 1 cc. of either is equivalent to 1 mg. of acetone. They are the 
solutions described by Shaffer (1908-09). 

The thiosulfate solution was kept in a brown bottle connected 
with a burette by a siphon, and was protected from the air by 
tubes containing soda-lime. The day after it was prepared it was 
standardized in the usual way against an equivalent solution of 
pure potassium biiodate containing 3.362 gm. per liter. The 
strength of the thiosulfate solution remained unchanged for sev¬ 
eral months. The iodine solution was standardized against this, 
and restandardized from time to time, as its strength varied 
slightly even when kept in a colored bottle. 

From these stock solutions dilution was made, using calibrated 
glassware. Water from a Barnstead still, which has a device 
for boiling water to expel ammonia and other volatile substances, 
was used for this dilution, and for all other work with acetone 
(see also Ljungdahl, 1919). To make the dilute iodine solutions, 
enough postassium iodide was used to give a final concentration 


46 


Determination of Acetone 


of about 3 per cent, and the stock solution diluted to shy or 
of its original strength. These solutions are equivalent 
respectively to 0.1, 0.02, and 0.01 mg. of acetone. The thiosul¬ 
fate solutions of corresponding strength were made by diluting 
the stock solution with distilled water, and were found to tritrate 
correctly against the equivalent iodine or biiodate solutions. 
The strength of the iodine solutions remained unchanged for 2 or 
3 days; after that it often increased slightly, probably due to 
oxidation of the relatively large amounts of potassium iodide 
present. The thiosulfate solutions were permanent for from 24 
to 48 hours, but not for a longer time. No special precautions 


TABLE i. 

Effect of Strength of Acid and of Time on the Titration of Dilute Iodine 

Solutions. 


Iodine. 

Thiosulfate. 

Acid concentration 
(H 2 SO<). 

Titrated at 
once. 

Titrated 
after 30 min. 

Strength. 

Amount. 

Strength. 


cc. 



cc. 

cc. 

0.001 n 

10 

0.001 n 

0.5 n 

10.0 

10.8 

0.001 n 

10 

0.001 n 

0.25 n 

9.93 

9.97 

0.001 n 

10 

0.001 n 

Slight excess. 

9.97 

9.93 


Volume of each solution equals 50 cc. 


were taken in preparing these solutions, as it was found more 
convenient to make a freshly diluted solution of thiosulfate daily. 

In titrating with these dilute solutions certain precautions 
were necessary. All solutions of sodium hydroxide gave a slight 
blank, the size of which depended on the amount and grade of 
the sodium hydroxide used. It was found that this blank was 
constant if the alkaline iodine solutions were allowed to stand for 
10 minutes, and that it did not increase in J hour; if a good grade 
of sulfuric acid in not too great excess (a final excess of 0.25 n or 
less) was used for acidifying; and if the solution was titrated 
within 15 minutes after adding the acid. 2 Table I shows the 
effect of excess of acid on the titration of dilute iodine solutions. 

2 Some grades of sulfuric acid contain substances which react with iodine 
solutions, and it was found advisable to allow the acidified solution to 
stand for 5 minutes before titrating. If the sulfuric acid is boiled for 10 
minutes, this error is decreased. 















TABLE II. 

Effect of Volume and Time on the Titration of Alkaline Iodine Solutions. 


R. S. Hubbard 


a> 

a 

J3 

"3 

> 

6 

o 

o 

o 

tM 


O 

GO 


3 

m 

_o 

3 

H 


30 min. 

CC. 

19.72 

d 


a 

CC. 

*0 



<M O 


. 05 05 

3 

V) 


« 05 05 


r-l 

d 

00 00 

a 

. oo oo 

V • 

o 

“ 05 05 

CO 

H 

• 

o 

d 

00 

a 

o 

00 

*0 

r-H 


Cl (M t- 


. 05 05 CO 

a 

O'*. 

° o o o 

HIH 

H r-H 


05 

05 


o 

00 

00 


. 

o 


o 

a 

o • 

° C 5 

»o 

r-H 


CO 

05 00 
05* 05* 


ic 

CO 


05 


d 

30 min. 

c. 

9.95 

19.87 

9.10 

18.92 

a 




d 

lO o 

. 1-10 

> 

a 

05 05 

d 

*0 

T—1 

o 



o 



*o 


n m o «5 



. 05 CO 00 1C 


a 

o .... 

“ 05 05 05 05 


HH 

H r-H 


a> 

-3 

o 


Strength. 

O.OIn 

O.OIn 

O.OOIn 

O.OOIn 

-d 


d 

• o o iO o 

o 

g H (M pH 

a 


<: 


pC 


W) 

£ £ (N <M 

d 

<u 

Fh 

i—i i—i O O 

o o o o 

M 

o' o o' o' 


P 

o 

• pH 

-a> 

3 

r—H 

o 

m 

<D 

a 

• pH 

-d 

o 

• pH 

a> 

.P 

p < 

cJ 

P"! 

c3 

a) 

-P 

-M 

T) 

P 

o3 

nO 

a> 

no 

no 

cj 


o 

« 

w 


o 

o 

o 

o 

H 

4- 

W 

o 

cj 

£ 


bfl 


p 

o 


o 

in 

a> 

n3 

*E 

o 

(h 

no 

>> 

-a 

6 

o 

no 

O 

w 

bO 

P 

o 

u 

+5 

w 

c3 


• no 
o S 

° £ 

i"H O 


to stand as shown before acidifying. 







































48 


Determination of Acetone 


The volume of the solution also affects the value of the blank 
in certain cases. If 0.001 n iodine solutions are used, the volume 
of the solution must be kept practically constant; when 0.002 n 
solutions are used, the volume may vary from 50 to 100 cc., but 
should not be more dilute; with 0.01 n solutions the variation 
may be very much greater. The value of the blank is so small 
that the determination with the stock solutions is not affected 
(Table II). 

When small amounts of acetone were determined, it was found 
necessary to insure the addition of sufficient alkali. Table III 
gives some results obtained with a solution of acetone containing 


TABLE III. 

Effect of the Concentration of Alkali and of Time on the Formation of 
Iodoform. 


Stood 

alkaline. 

Acetone 

present. 

Approximate normality. 

0.2 n 
NaOH 
found. 

0.15 n 
NaOH 
found. 

0.12 N 
NaOH 
found. 

0.10 N 
NaOH 
found. 

0.07 n 
NaOH 
found. 

0.04 n 
NaOH 
found. 

min. 

mg. 

mg. 

mg. 

mg. 

mg. 

mg. 

mg. 

5 

0.450 

0.450 

0.395 


0.443 



7 

0.450 





0.415 





(0.452 





10 

0.450 

0.438 

\ 0.439 

0.448 

0.447 

0.439 

0.382 




(0.445 





15 

0.450 

0.449 



0.455 



20 

0.450 

0.448 

0.452 


0.435 

0.448 


30 

0.450 


0.455 






10 cc. of 0.01 n iodine used in this determination in each experiment. 


about 0.5 mg. A final concentration of 0.1 n to 0.2 n sodium 
hydroxide gave complete formation of iodoform in 10 minutes, 
while smaller concentrations did not give complete formation 
within that time. The limitations described above and illus¬ 
trated in Tables I to III were the only limitations found to the 
application of the method. 

i Method . 

To acetone, contained- in a volume of 50 to 100 cc., a known 
amount of iodine was added. If there was probably very little 
acetone present—such amounts as are obtained from a few cc. 
















R. S. Hubbard 


49 


of normal blood or urine—10 to 25 cc. of 0.00207 n (1 cc. = 0.02 
mg. of acetone) iodine, diluted as described, were used; for 
amounts of acetone from 0.2 to 2.0 mg., such as are obtained from 
blood or breath of patients with a marked acetonemia, 25 cc. of a 
solution five times as strong were added; for larger amounts, 
such as are found in the urine of patients with marked acetonemia, 
the stock solutions as described by Shaffer (1908-09) were used. 
When amounts of acetone larger than 0.2 mg. were present it 
was found that the volume of solution in which the acetone was 
contained had little effect on the determination, and if several 
milligrams were present the reaction could be carried out in 
300 cc., as accurately as in 50 cc. No matter what amount of 
acetone is present, enough iodine must be used to give a distinct 
excess of the reagent. 

To the solution containing acetone and iodine, 2 cc. of a sodium 
hydroxide solution, v made by dissolving 200 gm. of sodium hydrox¬ 
ide in 300 cc. of water, were added, and the solution was shaken 
well for a few seconds. Long shaking was not found necessary. 
After the solution had stood alkaline for 10 minutes or more, 1 
to 2 cc. of sulfuric acid (1 part of concentrated acid to 1 part of 
water) were added, and the solution was titrated with 0.001, 0.01, 
or 0.1 n sodium thiosulfate. A little clear dilute starch solution 
was added before the end of the titration to serve as indicator. 
A blank was run every day to test the relative strength of the 
iodine and thiosulfate solutions, and the result calculated from 
the difference in value between the titration of this blank and 
that of the solution containing acetone. As stated above, dilute 
solutions of sodium thiosulfate were prepared daily. 

Acetone solutions used were carefully purified by redistilling 
repeatedly from fused calcium chloride until the product boiled 
sharply at 56°C. Samples of this product, when weighed out in 
small pipettes, sealed, the seal broken under water, and the 
acetone solution made to volume after the method of Marriott 
(1913-14 a), gave values on titration that agreed with the theo¬ 
retical ones. The samples analyzed were of the magnitude of 
20 mg. Solutions of this pure acetone were diluted to appro¬ 
priate strength, and were used for the determination of minute 
amounts of acetone. It was necessary to restandardize the 
strong solutions frequently, as all acetone solutions lose strength 
readily. 


THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. XL1II, NO. 1 


50 


Determination of Acetone 


Tables IV and V give results obtained by the method described. 
The figures in the column headed “Present” correspond with 
figures calculated from parallel determinations carried out on 
the strong acetone solutions from which the dilute solutions used 
were prepared. 

Since it was desired to test the effect of repeated distillation 
from various solutions upon acetone, a few experiments were 
carried out on the loss of acetone when exposed to the air under 

TABLE IV. 


Results on Solutions of Pure Acetone. 


Solutions used. 

Present. 

Found. 


mg. 

mg. 

0.00207 n iodine and 0.001035 n thiosulfate. 

0.0046 

0.0049 


0.00916 

0.0104 


0.0229 

0.0227 


0.0458 

0.0458 


0.0916 

0.0900 

- 

0.2290 

0.2345 

0.01035 n iodine and 0.01035 n thiosulfate. 

0.045 

0.038 


0.090 

0.082 


0.225 

0.218 


0.450 

0.450 


0.900 

0.887 


2.250 

2.235 


various conditions. It appears from the table that the percent¬ 
age loss of acetone under constant conditions varies with the 
volume of the solution used, but is practically independent of 
the amount of acetone present. The loss varies with the amount i 
of surface of the solution and with temperature, as was expected, 
but, if the volume of solution is 100 cc., there is very little more 
loss at 37°C. than at 20°C. (see Table V). If solutions were 
sealed there was no change in acetone content of jars exposed to 
room temperature for 2 hours. 







R. S. Hubbard 


51 


TABLE V. 


Loss of Acetone on Standing. 


Vol¬ 

ume. 

Ace¬ 

tone. 

Time of standing. 

Notes. 

f hr. 

1 hr. 

2 hrs. 

cc. 

mg. 

mg. 

per cent 

mg. 

per cent 

mg. 

per cent 


50 

7.15 

6.79 

95 

6.49 

91 

5.69 

80 

In 200 cc. Erlen- 

50 

0.674 

0.621 

92 

0.601 

89 

0.546 

81 

meyer flasks. 

100 

7.15 

7.06 

99 

6.95 

97 

6.58 

92 


100 

0.674 

0.663 

99 

0.653 

97 

0.628 

93 


50 

6.43 

5.82 

91 

5.47 

85 

4.87 

76 

In small specimen 

100 

6.43 

6.12 

95 

5.92 

92 

5.52 

86 

j ars, diameter 6 

150 

6.43 

6.27 

98 

6.04 

94 

5.72 

89 

cm. 

50 

6.43 

3.85 

60 

3.30 

51 

1.75 

27 

In large specimen 

100 

6.43 

4.70 

73 

4.40 

68 

2.85 

44 

jars, diameter 16 

150 

6.43 

4.98 

77 

4.85 

75 

3.50 

55 

cm. 

50 

8.15 





6.20 

76 

Small specimen jars 

50 

20.60 





15.75 

76 

at room tempera¬ 

100 

8.15 

7.65 

94 

7.20 

88 

6.70 

82 

ture, 20 °C. 

100 

20.60 

19.42 

94 

18.60 

90 

17.17 

83 


50 

8.15 



6.10 

75 



Small specimen j ars 

50 

20.60 



14.95 

73 



heated to 38°C. in 

100 

8.15 



7.20 

88 



incubator. 

100 

20.60 



17.98 

87 





Distillation of Acetone from Various Oxidizing Reagents. 

In order to separate acetone from various other compounds 
which also react with ’ alkaline iodine solutions, it may conven¬ 
iently be distilled from various reagents. Table VI gives 
results obtained by distilling acetone solutions from sodium 
peroxide; sulfuric acid plus potassium permanganate; sulfuric 
acid plus potassium dichromate; and by repeated distillation 
from sodium peroxide. The amount of acetone under the heading 
“Control” was measured and titrated simultaneously with the 
specimen oxidized. Table VI shows that acetone is not oxidized, 
under the conditions described, by sodium peroxide or by sulfuric 
acid plus potassium dichromate, or by sulfuric acid plus potassium 
















52 Determination of Acetone 

permanganate if the concentrations of acid and permanganate 
are properly regulated; if the volume is about 150 cc. and 
the concentration of sulfuric acid about 0.5 n, and 0.2 gm. of 
potassium permanganate is added, from 0.07 to 20 mg. of ace- 

TABLE VI. 


Distillation of Acetone from Oxidizing Reagents. 


From Na202.* 

From H2SO4 + KMnC>4. 


c 








Notes. 

O 

% 

Control. 

Found. 

Vol¬ 

ume. 

02 

W 

KMn04 

Control. 

Found. 


gm. 

mg. 

mg. 

cc. 

cc. 

gm. 

mg. 

mg 





150 

25 

Few 

7.68 

7.70 







crystals. 




0.25 

6.90 

6.70 

150 

25 

0.1 

7.70 

7.40 


0.25 

6.78 

6.80 

150 

25 

0.2 

7.70 

6.90 


0.25 

6.70 

6.75 

150 

10 

0.1 

19.82 

19.60 

H 2 SO 4 is about 

0.5 

0.0757 

0.0749 

150 

10 

0.2 

19.87 

19.17 

20 Xn. Made 

0.5 

3.64 

3.70 

150 

5 

0.2 

0.0757 

0.0763 

by diluting 1 

0.5 

6.85 

6.85 

150 

5 

0.2 

0.682 

0.682 

part H 2 SO 4 

0.5 

17.38 

17.25 

150 

5 

0.2 

19.82 

19.60 

with 1 part 

0.5 

18.60 

18.32 

150 

5 

0.3 

7.05 

6.92 

water. 

0.5 

35.80 

36.15 

100 

10 

0.1 

7.85 

7.65 


1.0 

6.73 

6.55 

100 

5 

0.2 

20.02 

19.54 




6.78 







1 .U 

O. (O 







1.0 

14.05 

14.10 


From H2SO4 + K2Cr2C>7. 



2.0 

7.10 

6.25 


O 





2.0 

6.83 

6.97 

Vol¬ 

ume. 

CO 

W 

K 2 Cr 2 C >7 

Control. 

Found. 





cc. 

cc. 

gm. 

mg. 

mg. 





150 

25 

1 

0.816 

0.827 





150 

25 

1 

7.60 

7.67 





150 

25 

1 

19.95 

19.80 





100 

25 

1 

7.60 

7.53 




i 

£ 

Control. 

Found. 


gm. 

mg. 

mg. 

Distilled from Na 2 C >2 and redistilled three times from 

0.5 

0.605 

0.610 

the same amount of the reagent. 

0.5 

5.60 

5.20 


1.0 

6.88 

6.85 


1.0 

6.72 

7 07 


♦Volume of 100 cc. 












































R. S. Hubbard 


53 


tone may be recovered quantatively. If much greater concen¬ 
trations of acid and permanganate are used, there is some loss 
of acetone, but the method can be applied safely to the separa¬ 
tion of acetone from substances which are more easily oxidized 
by sulfuric acid-permanganate solution than acetone is. 

Table VII shows more in detail the effect of prolonged exposure 
to some of these oxidizing reagents. A known amount of acetone 
was added to each of several flasks, with water to the volume 
indicated. Oxidizing reagents were then added, the flask was 
connected with a condenser and receiver, and placed on a boiling 
water bath. After the solution had stood in boiling water for 
from 10 to 30 minutes, the water bath was removed without 
disconnecting the flask, and the contents of the flask were dis¬ 
tilled actively for 10 minutes. The acetone in the receiving 
flask was then titrated by the method described. Not more 
than 10 per cent of the acetone used was found in the receiving 
flask when an alkaline, acid, or neutral solution was heated on 
a boiling water bath for 30 minutes connected in this way. Con¬ 
trols were run as in the preceding experiments. 

Table VII shows that sodium peroxide, except in very large 
amounts, does not oxidize acetone, nor do the concentrations of 
sulfuric acid and potassium dichromate tested. The table 
brings out more clearly than does the preceding one the effect of 
concentration of sulfuric acid and potassium permanganate in 
independently increasing oxidation. The concentrations of these 
two substances which gave no oxidation on distillation gave 
oxidation when heated under these conditions. 

Table VIII presents the effect of the following treatment. 
Acetone was distilled from acid and redistilled from alkali to a 
volume of about 150 cc. To this distillate were added 5 cc. of 
sulfuric acid diluted with 1 part of water and 0.2 gm. of potassium 
permanganate; this was distilled to give a volume of about 100 
cc. in the distillate. To this distillate was added 0.5 gm. of 
sodium peroxide and this was again distilled. This distillate was 
titrated by the method described. 

The following substances treated as described above gave no 
reaction with alkaline iodine solutions: 10 cc. of saturated chloro¬ 
form, gasoline, benzene, toluene solutions; 500 mg. of phenol, 
methyl alcohol, formaldehyde; about 400 mg. of ethyl alcohol 



54 


Determination of Acetone 


TABLE VII. 

Oxidation of Acetone on Boiling Water Bath,. 
Sodium peroxide. 


Heated 10 min. on water bath. 


Heated 30 min. on water bath. 


Acetone. 

Volume. 

NazCb 

Found. 

Acetone. 

Vol- 

Na20a 

Found. 

Initial. 

Controls. 

Initial. 

Controls. 

ume. 

mg. 

mg. 

cc. 

gm. 

mg. 

mg. 

mg. 

cc. 

gm. 

mg. 



100 

0 

6.55 



100 

0 

6.80 



100 

0.25 

6.62 

7.20 

6.68 

100 

0.25 

6.68 

7.12 

6.54 

100 

0.50 

6.82 

6.80 

100 

0.50 

6.80 

6.70 

100 

1.00 

6.42 



100 

1.00 

6.75 



100 

2.00 

5.94 








75 

0.50 

6.62 

7.12 

6.60 

100 

2.00 

6.05 



150 

0.50 

6.64 

6.40 





Potassium permanganate and sulfuric acid.* 


Acetone. 

Volume. 

Time of 

Acid. 

KMnOi 

Found. 

Initial. 

Controls. 

heating. 

mg. 

mg. 

cc. * 

min. 

cc. 

gm. 

mg. 



150 

10 

0 

0.1 

6.59 



150 

10 

5 

0.1 

6.27 



150 

10 

5 

0.2 

5.92 

7.17 

6.57 

150 

10 

10 

0.1 

• 6.19 

6.57 

150 

10 

10 

0.2 

5.49 



150 

10 

10 

Few crystals. 

6.97 



150 

10 

25 

u u 

7.07 



150 

10 

25 

0.1 

5.37 

7.10 

6.91 

150 

30 

5 

0.1 

6.08 


Potassium dichromate and sulfuric acid.* 


Acetone. 

Volume. 

Time of 

Acid. 

I&CnO? 

Found. 

Initial. 

Controls. 

heating. 

mg. 

mg. 

cc. 

min. 

cc. 

gm. 

mg. 

7.87 

7.57 

150 

© 

CO 

25 

1 

7.55 

7.62 

150 

30 

25 

1 

7.75 


* Sulfuric acid is about 20 Xn. 


























































R. S. Hubbard 


55 


(probably partially removed by the repeated fractional distilla¬ 
tion (Richter-Quittner, 1919) and partially by oxidation), 50 
mg. of acetaldehyde, and 50 mg. of ether. 3 Various normal 
urines and bloods have been analyzed by this technique, and they 
gave values of from 0.01 to 0.03 mg. of acetone in the 10 cc. 
samples taken. 4 

TABLE VIII. 


Successive Distillation of Acetone from Different Reagents . 


Reagents used. 

Control. 

Found. 


mg. 

mg. 

Distilled successively from H 2 S0 4 , NaOH, 

0.264 

0.248 

H 2 S0 4 +KMn0 4 , and Na 2 0 2 . 

0.664 

0.669 

Control measured and titrated when the solu¬ 

0.664 

0.661 

tion was. It was exposed to the air during 

1.303 

1.361 

oxidation and distillation of the solution 

1.605 

1.608 

studied. 

3.44 

18.05 

3.44 

17.95 


CONCLUSION. 

A method has been described by which the Messinger titration' 
method may be applied to dilute acetone solutions, certain 
reactions of acetone have been studied, and a method has been 
proposed by which small amounts of acetone can be separated 
from much larger amounts of alcohol and other interfering com¬ 
pounds. 

My thanks are due to Dr. Philip A. Shaffer for his advice and 
assistance in this work. 

3 Ether is oxidized by acid permanganate solutions as it is by dichromate 
solutions to give a compound which reacts like acetone (Short, 1920). If 
the ether present is less than 50 mg., this compound is removed by the 
subsequent treatment with sodium peroxide. 

4 A paper in which this technique is applied to normal urine and blood is 
in preparation. 









56 


Determination of Acetone 


BIBLIOGKAPHY. 

Bang, I., Biochem. Z., 1913, xlix, 19. 

Collischonn, F., Z. anal. Chem., 1890, xxix, 562. 

Csonka, F. A., J. Biol. Chem., 1916, xxvii, 209. 

Deniges, G., Compt. rend. Acad., 1898, cxxvi, 1868; cxxvii, 963. 

Engfeldt, N. O., Z. physiol. Chem., 1915, xcv, 337; Berl. klin. Woch., 1915, 
lii, 796. 

Folin, 0., and Denis, W., J. Biol. Chem., 1914, xviii, 263. 

Frommer, V., Berl. klin. Woch., 1905, xlii, 1008. 

Geelmuyden, H. C., Z. anal. Chem., 1896, xxxv, 503. 

Hubbard, R. S., J. Biol. Chem., 1917, xxix, p. xiv. 

Kramer, G., Ber. chem. Ges., 1880, xiii, 1000. 

Lenk, E., Biochem. Z., 1916, lxxviii, 224; abstracted in Chem. Abst., 1917, 
xi, 823. 

Ljungdahl, M., Biochem. Z., 1917, lxxxiii, 103. 

Ljungdahl, M., Biochem. Z., 1919, xciii, 325. 

Marriott, W. McK., J. Biol. Chem., 1913-14, a, xvi, 281; b, 289. 

Marsh, J. E., and Fleming-Struthers, R. de J., J. Chem. Soc., 1905, lxxxvii, 
1878. 

Messinger, J., Ber. chem. Ges., 1888, xxi, 3366. 

Oppenheimer, C., Berl. klin. Woch., 1899, xxxvi, 828. 

Personne, J., Compt. rend. Acad., 1863, lvi, 951. 

Richter-Quittner, M., Biochem. Z., 1919, xciii, 163. 

Sammett, O., Z. physiol. Chem., 1913, lxxxiii, 212. 

Scott-Wilson, H., J. Physiol., 1911, xlii, 444. 

Shaffer, P. A., J. Biol. Chem., 1908-09, v, 214. 

Short, J. J., J. Biol. Chem., 1920, xli, 503. 

Treadwell, F. P., translated by Hall, W. T., Analytical Chemistry, New 
York and London, 4th edition, 1915, ii, 652. 

Van Slyke, D. D., J. Biol. Chem., 1917, xxxii, 455. 



Keprinted from The Journal of Biological Chemistry, Vol. XLIII, No. 1, 1920 


DETERMINATION OF ACETONE IN EXPIRED AIR.* 

By ROGER S. HUBBARD. 

{From the Laboratory of the Clifton Springs Sanitarium, Clifton Springs.) 

(Received for publication, June 1, 1920.) 

The fact that acetone is present in large amounts in the breath 
of patients suffering from severe.diabetes has been known for a 
long time, and the high percentage of acetone which might occur 
in the breath was demonstrated by Geelmuyden in 1897. He 
injected acetone into rabbits, and showed that as much as 75 
per cent of the acetone recovered might be found in the breath. 
In spite of these facts there are comparatively few quantitative 
determinations of acetone recorded in the literature, and almost 
all of these are in severe cases of diabetes. 

One of the reasons for this is a lack of a convenient method for 
determining such small amounts of acetone as are found in the 
breath of normal subjects, or of subjects suffering from a moder- 
rate degree of acetonemia. Nebelthau (1897) had patients 
suffering from diabetes breathe directly through alkaline iodine 
solutions, after which he determined the amount of acetone by 
the Messinger (1888) titration. He admitted that this method 
was inexact, due to loss of iodine. Such a method can, however, 
be used to show the presence of acetone in breath, and has been 
adapted for its determination when present in large amounts by 
Marriott (1914), and for the determination of acetone in the air 
in chemical plants by Elliott and Dalton (1919). Scott-Wilson 
(1911) used the mercuric cyanide reagent described by him to 
demonstrate the presence of acetone in the breath of patients 
suffering from “ acidosis,” but did not describe a technique for 
a quantitative method. Geelmuyden (1897) described a method 

* A preliminary report of this work was given before the meeting of the 
American Society of Biological Chemists in Baltimore in April, 1919. 

Part of the work was carried out in the chemical laboratory of Harvard 
University through the courtesy of Dr. L. J. Henderson in February, 1919. 

57 


58 


Acetone in Expired Air 


in which air from the lungs was oxidized in a combustion 
furnace, and the acetone calculated from the carbon dioxide 
so produced. He experimented with small animals in a Petten- 
kofer-Yoit calorimeter. The air, drawn from the chamber through 
sodium hydroxide, passed to the furnace, where it was oxidized, 
and the carbon dioxide so obtained was caught in a second 
series of bottles of sodium hydroxide. Muller (1897-98) devel¬ 
oped a technique which might be used with human subjects. 
He caused the patient to breath through a series of four Woulff 
bottles containing 250 to 500 cc. of water kept cold by ice. The 
pressure of the water in the bottles was reduced by a pump 
capable of moving a large volume of air, and subjects could 
breathe for J to 1 hour without discomfort. Acetone in the 
bottles was determined by the Messinger titration. This tech¬ 
nique included in the determination other substances which react 
with alkaline iodine solutions and which may be present in the 
breath. Voit (1899) used this method of Muller, in connection 
with the Pettenkofer-Voit respiration chamber, to determine 
the effect of diet upon the excretion of acetone by small animals. 

Folin and Denis (1915), in a study of the metabolism of two 
fasting women, have used a method based on the retention of 
acetone by a sodium bisulfite solution, and its subsequent deter¬ 
mination with Scott-Wilson reagent, as described by them (Folin 
and Denis, 1914). Their patients breathed for 3 minutes 
through 10 cc. of 0.5 per cent sodium bisulfite solution. The 
results showed a marked increase of acetone during fasting, but 
did not show acetone in the breath of these patients on a normal 
diet. The method described below resembles this one in many 
essential features. 

Folin and Denis (1914) have stated that acetone is retained 
almost as completely when blown through a 2 per cent solution 
of sodium bisulfite as is ammonia by a solution of hydrochloric 
acid. Preliminary tests showed that acetone in a stream of 
carbon dioxide from a Kipp generator was quantitatively retained 
by such a solution in spite of the decomposition of bisulfite. 

Experiments were also made on the recovery of acetone added 
to sodium bisulfite solutions after distillation from sulfuric acid 
plus potassium permanganate and from sodium peroxide suc¬ 
cessively, a procedure which separates acetone from rather large 


R. S. Hubbard 


59 


amounts of alcohol, acetaldehyde, and other interfering com¬ 
pounds. 1 To known amounts of acetone plus 2 gm. of sodium 
bisulfite in about 150 cc. of water, 10 cc. of 10 per cent sodium 
hydroxide were added, and the solution was distilled. The 
distillate was redistilled successively from sulfuric acid plus 
potassium permanganate and from sodium peroxide. Table I 
shows the results of such experiments. The maximum loss of 
acetone was about 6 per cent, and was not due to oxidation, as 
the same loss was shown by the control solutions exposed to the 
air. 


TABLE i. 

Recovery of Acetone Added to Bisulfite Solutions. 


Description. 

Acetone 

added. 

Acetone 

control. 

Thiosulfate. 

Acetone 

recov¬ 

ered. 


mg. 

mg. 


CC. 

mg. 

2 gm. of NaHS0 3 +acetone as shown, 

0.0 



0.57 

0.011 

distilled from NaOH, and redis¬ 

0.0 



0.88 

0.018 

tilled successively from H2SO4+ 

0.0 


0 . 002 n * 

.1.10 

0.022 

KMn0 4 , and from Na 2 0 2 as de¬ 

0.0 



0.45 

0.009 

scribed. Control column gives 

0.0 



0.70 

0.014 

results from acetone in about 100 

0.0 



0.56 

0.011 

cc. of water exposed to the air 
during the experiment. First six 

0.096 

0.087 

0 . 002 n * 

5.01 f 

0.102 

results show blanks by this 

0.685 

0.670 

0 . 01 n * 

6.67f 

0.667 

method. 

7.40 

7.02 

0 . 1 n * 

6.92f 

6.92 


7.40 

6.82 

0 . 1 n * 

7.12f 

7.12 



17.50 

0 . In * 

17.18f 

17.18 


* Approximate normality, true figure 103.47 per cent of these, 
f Corrected for the blank given by the reagents. 


Method. 

For determining acetone in air, 75 cc. of a freshly prepared 2.5 
per cent sodium bisulfite solution were measured into each of two 
bottles. The patient breathed through a mask (a celluloid ether¬ 
izing mask with the valve removed), or through a mouthpiece. 
He received air through a simple valve placed in a vertical posi¬ 
tion to insure complete closure with expiration. The glass 
tubing used was 9.5 mm. and the bottles were 75 mm. in diameter. 
The amount of glass and rubber tubing was reduced to a minimum. 

1 Hubbard, R. S., J. Biol. Chem., 1920, xliii, 43. 













60 


Acetone in Expired Air 


After the subject had breathed for 5 or 10 minutes (usually 
for 10 minutes) the bottles were disconnected, 10 cc. of 10 per 
cent sodium hydroxide were added to each, and the contents of 
each were washed separately into a 500 cc. Kjeldahl flask and 
treated as described below to remove interfering compounds 
already referred to. The solution was first distilled for 10 min¬ 
utes through a water-cooled condenser into a second Kjeldahl 
flask containing enough water to cover the end of the receiving 
tube (Marriott, 1913-14 a) to give a final volume of about 150 cc. 
To the contents of the receiving flask 5 cc. of sulfuric acid (one 
part of concentrated acid plus one part of water) and about 0.2 
gm. of potassium permanganate were added. The solution was 
then redistilled with the same precautions to give a volume of 
about 100 cc. in a third distilling flask. To this flask 0.5 gm. of 
sodium peroxide was added, and a final distillation made. This 
final distillation was made into a little distilled water to give a 
volume of between 50 and 100 cc., and the acetone determined 
by the technique described in the preceding paper, 1 or it was 
made into 25 cc. of Scott-Wilson reagent, 2 and the acetone esti¬ 
mated by the degree of turbidity produced. 

If the latter method was used, standards containing approxi¬ 
mately the same amount of acetone were distilled into the same 
amount of reagent; all solutions were made to 100 cc., and the 
turbidities produced were compared in Nessler tubes. By this 
method readings could be made within 0.005 mg. For more 
exact determinations, solutions were compared with the nearest 
standard either in a nephelometer (Marriott, 1913-14 b), or, if 
more than 0.2 mg. of acetone was present, in a colorimeter accord¬ 
ing to the technique of Folin and Denis (1914). 

Usually slight blanks were obtained whether the acetone was 
determined by the Messinger titration or by the turbidity method. 
Blanks by the iodine method are given in Table I, blanks by the 
turbidity method were usually about 0.007 mg., and were prob- 

2 Scott-Wilson’s reagent may be prepared as follows (Marriott, 1913-14 
a, p. 284, foot-note): “Mercuric cyanide, 10 grams; Sodium hydroxide, 180 
grams; Water, 1200 cc. The solution is agitated in a flask and 400 cc. of a 
0.7268 per cent solution of silver nitrate slowly run in.” The solution 
should be prepared at least 24 hours before it is used, and should be filtered 
if it is not perfectly clear. 


R. S. Hubbard 


61 


ably produced by a trace of ammonia. The source of the blanks 
found with the iodine method was in the distillation from sodium 
peroxide, and probably arose from oxidation of some compound 
(possibly fat) which produced a substance that reacted with alka¬ 
line iodine solutions. To keep this blank constant, the flasks 
used for the last distillation must be kept free from grease, and 
the amount and approximate concentration of sodium peroxide 
must be kept fairly constant. Table II gives results on a series 
of normals in which the two methods are compared. The values 
are corrected for the blanks. 


TABLE II. 

Comparison of Results by Iodine and Turbidity Methods. 


Subject. 

Turbidity method. 
Found. 

Iodine method. 
Found. 


mg. 

mg. 

R. S. H. 

0.035 

0.040 

M. L. 

0.015 

0.015 

Mrs. S. 

0.007 

0.007 

Dr. O. 

0.025 

0.025 

N. B.:. 

0.025 

0.025 


Each subject breathed for 10 min. in both tests. 


To find out whether the oxidation by permanganate and sodium 
peroxide caused any change in the value obtained, samples of 
breath collected as described were distilled from alkali and acid 
only, to remove sulfurous acid and ammonia, and other samples 
of breath, obtained as nearly simultaneously as possible, were 
analyzed by oxidation. By iodine titration values as muclras 
50 per cent lower were obtained after oxidation; by the turbid¬ 
ity method, results after oxidation agreed with those obtained 
after distillation within 0.005 mg. There is, therefore, in normal 
breath a substance, not acetone, which reacts with alkaline iodine 
solutions, and which is removed by oxidation with acid perman¬ 
ganate and sodium peroxide. This substance does not react 
with Scott-Wilson’s reagent. The agreement of the results 
obtained by Scott-Wilson’s reagent after oxidation with results 
after distillation shows an absence in the breaths studied of appre¬ 
ciable amounts of secondary propyl alcohol, or other compounds 












62 Acetone in Expired Air 

which can give acetone on oxidation with potassium perman¬ 
ganate. 

To show the completeness with which acetone can be recovered 
from the breath by the apparatus described, two experiments 
were run. First, a normal subject breathed through three 
bottles of 2.5 per cent sodium bisulfite instead of two, and the 
contents of the third bottle were analyzed with negative results; 
second, a bottle containing a known amount of acetone in 75 cc. 
of water was placed between the valve and the first bottle con¬ 
taining bisulfite. Table III gives a record of several such experi¬ 
ments, and shows a high percentage of recovery of relatively 
large amounts of acetone. 


TABLE III. 


Recovery of Acetone Added to Breath. 


Description. 

Pres¬ 

ent. 

Resi¬ 

due. 

1st 

bottle. 

2nd 

bottle. 

Total 

recov¬ 

ered. 


mg. 

mg. 

mg. 

mg. 

mg. 

Breathed 10 min. through bottle con¬ 

3.88 

1.43 

0.98 

0.83 

3.24 

taining known amount of acetone into 

3.88 

1.66 

1.40 

0.41 

3.47 

two bottles containing NaHSOs solu¬ 

2.65* 

0.75 

1.25 

0.70 

2.70 

tion. 

2.65* 

0.30 

1.70 

0.50 

2.50 


2.65* 

0.30 

1.65 

0.45 

2.40 


1.50 

0.78 

0.51 

0.28 

1.57 


* Experiments in which acetone was warmed to 50°C. 


To prove the efficiency of the mask, a bottle containing 75 cc. 
of 2.5 per cent sodium bisulfite was connected with the Benedict 
portable respiration apparatus (a closed circuit respiration sys¬ 
tem) (Benedict, 1918) in such a way that the air from the subject 
passed through this bottle before it passed through the bottle of 
soda-lime used to absorb C0 2 . By this arrangement sulfur diox¬ 
ide was absorbed, and did not reach the subject’s mouth. Pre¬ 
liminary tests showed that as much as 5 mg. of acetone intro¬ 
duced into the machine were recovered quantitatively. Com¬ 
parative tests using this calorimeter, and the mask and two bottles, 
gave the same value for acetone within 0.002 mg. Experiments 
were run to determine whether the presence of this bottle of 














R. S. Hubbard 


63 


sodium bisulfite had any effect on the determination of meta¬ 
bolism by the apparatus, and showed that there was no such 
effect. 

Table IY shows the results of the method on a series of normal 
subjects. These results were obtained by using the face mask, 
and analyzing with Scott-Wilson’s reagent after oxidation with 

TABLE IV.' 


Results of Turbidity Method on Normal Subjects. 


No. and sex. 

1st bottle. 

2nd bottle. 

Total 

determination. 

Per hour. 


mg. 

mg. 

mg. 

mg. 

4c? 

0.068 

0.013 

0.081 

0.49 

59 

0.018 

o.oii 

0.029 

0.17 

69 

0.018 

0.008 

0.026 

0.16 

9c? 

0.042 

0.013 

0.055 

0.33 

10 c? 

0.033 

0.010 

0.043 

0.26 

119 

0.033 

0.010 

0.043 

0.26 

13c? 

0.015 

0.008 

0.023 

0.14 

14 9 

0.038 

0.010 

0.048 

0.29 

15 9 

0.038 

0.010 

0.048 

0.29 

16c? 

0.023 

0.008 

0.031 

0.19 

18 9 

0.028 

0.008 

0.036 

0.22 

21 c? 

0.023 

0.010 

0.033 

0.20 

22 c? 

0.038 

0.018 

0.056 

0.34 

23 9 

0.023 

0.005 

0.028 

0.17 

249 

0.023 

0.010 

0.033 

0.20 

25 c? 

0.028 

0.008 

0.036 

0.22 

28 9 

0.028 

0.011 

0.039 

0.23 

35 9 

0.028 

0.005 

0.033 

0.20 

37 c? 

0.023 

0.003 

0.026 

0.16 

38 9 

0.028 

0.013 

0.041 

0.25 

40 c? 

0.068 

0.018 

0.086 

0.52 

42 9 

0.053 

0.028 

0.081 

0.49 


Each subject breathed 10 min. 


potassium permanganate and sodium peroxide. The results are 
corrected for the slight blank given by the reagents. 

Table V shows the agreement which was found between dupli¬ 
cates, when the subject breathed for different periods within the 
same hour, and the different results obtained on the same subject 
on different days. A few results on cases showing an acidosis 
are included. 












64 


Acetone in Expired Air 


TABLE V. 

Acetone in Breath. 


o 

& 

Subject. 

Date. 

Breathed. 

1st bottle. 

2nd bottle. 

Total. 

Per hour. 

Remarks. 



1919 

min. 

mg. 

mg. 

mg. 

mg. 


3 

Ad' 

Apr. 15 

10 

0.028 

0.013 

0.041 

0.25 

Normal subject. 

33 


tt 

19 

10 

0.063 

0.018 

0.081 

0.49\ 

Nos. 33 and 34 determined 

34 


tt 

19 

5 

0.038 

0.011 

0.049 

0.59] 

in the same hour. 

39 


tt 

20 

10 

0.038 

0.023 

0.067 

0.37 


1 

Bd 

tt 

12 

10 

0.03 

0.01 

0.040 

0.24 

Normal subject. 

2 


tt 

12 

10 

0.025 

0.005 

0.030 

0.18 


29 


tt 

16 

2 

0.023 

0.005 

0.028 

0.84] 

Nos. 29, 30, and 31 done 

30 


tt 

16 

10 

0.112 

0.028 

0.140 

0.84> 

within the same hour. 

31 


it 

16 

5 

0.058 

0.018 

0.076 

0 .91 J 


32 


tt 

19 

10 

0.063 

0.023 

0.086 

0.52 


17 

C 9 

tt 

16 

5 

0.193 

0.020 

0.213 

2.6 

Diabetic; Legal positive.* 

19 

D 9 

It 

16 

5 

0.010 

0.003 

0.013 

0.16 

“ “ negative.* 

20 

E 9 

tt 

16 

5 

0.058 

0.023 

0.081 

0.97 

“ “ positive.* 

43 

Fd 

tt 

21 

5 

1.113 

0.133 

1.25 

15.0 

Exophthalmic goiter. 

44 

Gd 

It 

22 

10 

0.713 

0.164 

0.88 

5.24 

Diabetic; Legal positive.* 


* Legal’s reaction done on urine at the same time that the breath sample 
was obtained. 


SUMMARY. 

In the paper given above a convenient method is described by 
which acetone can be determined in the expired air by iodine 
titration or by precipitation with Scott-Wilson’s reagent after 
removal of such interfering compounds as primary alcohols, 
aldehydes, phenols, and the simple cyclic hydrocarbons. Close 
agreement of results obtained by these chemically different 
methods of determination on the breath of normal subjects 
indicates strongly that the compound so determined is acetone. 
A series of results obtained by the method on normal subjects 
and on a few pathological cases is included. 




















R. S. Hubbard 


65 


BIBLIOGRAPHY. 

Benedict, F. G., Boston Med. and Surg. J., 1918, clxxviii, 667. 
Elliott and Dalton, J., Analyst, 1919, xliv, 132. 

Folin, O., and Denis, W., J. Biol. Chem., 1914, xviii, 263. 

Folin, O., and Denis, W., J. Biol. Chem., 1915, xxi, 183. 
Geelmuyden, H. C., Z. physiol. Chem., 1897, xxiii, 431. 
Marriott, W. McK., J. Biol. Chem., 1913-14, a, xvi, 281; b, 289. 
Marriott, W. McK., J. Biol. Chem., 1914, xviii, 516. 

Messinger, J., Ber. chem. Ges., 1888, xxi, 3366. 

Muller, J., Arch. Exp. Path. u. Pharmacol., 1897-98, xl, 351. 
Nebelthau, A., Zentr. inn. Med., 1897, 977. 

Scott-Wilson, H., J. Physiol., 1911, xlii, 444. 

Voit, F., Deutsch. Arch. klin. Med., 1899, lxvi, 564. 




/ 




THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. XL1H, NO. 




v .X* . • r . f :• • ■ 'i 













































Reprinted from The Journal of Biological Chemistry 
Vol. xlix, No. 2, December, 1921 


NOTE ON THE DETERMINATION OF /3-HYDROXY- 
BUTYRIC ACID.* 

By ROGER S. HUBBARD. 

{From the Laboratory of Biological Chemistry , Washington University 
School of Medicine, Saint Louis.) 

(Received for publication, October 4, 1921.) 

In studying the determination of /3-hydroxybutyric acid three 
methods have been most used. These are: first, the isolation of 
the acid, with its subsequent determination by the polariscope; 
second, the dehydration of the acid to give a-crotonic acid; and 
third, the oxidation of the acid to give acetone. 

In Hurtley’s (1915-16) paper, in which the discovery and 
subsequent investigation of the properties of this acid are dis¬ 
cussed exhaustively, it is shown that all of these methods are 
closely connected with the first studies made by Kulz (1884), 
Stadelmann (1883), and Minkowski (1884) into the properties of 
the organic acid isolated by them from urines which contained 
an excess of base greater than that theoretically needed to neu¬ 
tralize the inorganic acids found. 

Kulz (1884) first fermented diabetic urine and determined the 
rotation, thus measuring the amount of the acid present. Wolpe 
(1886) modified this method. He extracted the acidified urine 
with ether, took up the extracted acid with water, and deter¬ 
mined the acid in the extract by the polariscope. Magnus-Levy 
(1901), Bergell • (1901), Geelmuyden, and Black (1908-09) im¬ 
proved this extraction method further. Ohlsson (1916) has 
recommended ethyl acetate instead of ether for the extraction. 

The second method was suggested by Darmstaedter (1903). 
He converted /3-hydroxybutyric acid into a-crotonic acid by the 
action of concentrated sulfuric acid, and determined the unsatu- 

* This paper formed a part of a thesis presented in partial fulfilment 
of the requirements for the degree of Doctor of Philosophy at Washington 
University, Saint Louis, in June, 1921. 

351 


352 


/3-Hydroxybutyric Acid 


rated acid by titration after distillation—a reaction noted by 
Kulz (1884) and Stadelmann (1883). This method has been 
modified by Pribram (1911-12) and others, but has not been found 
to be very satisfactory (Shaffer, 1908-09; Shindo, 1907). 

The third method for the determination of /3-hydroxybutyric acid 
was proposed by Shaffer (1908-09) based on a reaction mentioned 
by Minkowski (1884). /3-Hydroxybutyric acid when oxidized 
with sulfuric acid and potassium dichromate forms acetone and 
carbon dioxide, probably giving acetoacetic acid as an inter¬ 
mediary product. In his first paper, Shaffer (1908-09) stated 
that this oxidation was quantitative. Embden and Schmitz 
(1910) in Abderhalden’s Handbuch stated their belief that the 
results by this method were low. In 1913, Shaffer and Marriott 
(1913-14) studied the reaction, using pure /3-hydroxybutyric acid 
prepared from calcium zinc /3-hydroxybutyrate. They found 
that their recovery was from 5 to 10 per cent low. The method 
described is a slow one, as 3 to 4 hours are required for maximum 
oxidation with the amounts of sulfuric acid and potassium dichro¬ 
mate recommended. Marriott (1914), Folin and Denis (1914), 
and Kennaway (1914) have used this oxidation of /3-hjrdroxy- 
butyric acid in connection with different methods for the 
determination of acetone. Lately Van Slyke (1917) has studied 
the reaction carefully and investigated the causes that lead to 
incomplete oxidation. At the time the experiments reported 
below were performed, only the preliminary report of this last 
paper was available. 

For determining /3-hydroxybutyric acid in urine the method of 
oxidizing with acid and potassium dichromate was chosen as most 
suitable. Two main problems presented themselves for solution 
in using this method. Was it possible to find some way in which 
the determination could be made exact? That is to say, could 
conditions of the determination be found that would lead to a 
recovery of the theoretical amount of acetone from a given 
amount of /3-hydroxybutyric acid? Could the time of oxidation 
be shortened? Many different oxidizing mixtures with widely 
varying amounts of sulfuric acid and potassium dichromate were 
tried, and large and small amounts of /3-hydroxybutyric acid 
were used in the determinations, but a recovery of the theoretical 
amount of acetone was not accomplished. In attempting to 


R. S. Hubbard 


353 


solve the second problem, an investigation of conditions govern¬ 
ing the oxidation of /3-hydroxybutyric acid by acid dichromate 
solutions was undertaken, and it was found that the rate and 
completeness of oxidation depend on the relative concentrations 
of acid and dichromate. Van Slyke (1917) has published a 
series of experiments establishing this fact, and the results found 
do not differ from those described by him. 

Shaffer and Marriott (1913-14) give the following description 
of the method (p. 271): “the contents of the distilling flask 
containing the oxybutyric acid was diluted to about 600 cc., 30 
cc. of sulphuric acid (sp. gr. 1.59) added, and a total of about 0.5 
gram of K 2 Cr 2 07 in very dilute solution dropped in during the 
distillation which was continued about three and one-half hours.” 

Using this method as a basis, two methods were worked out 
for the determination of /3-hydroxybutyric acid in a shorter period 
of time. The first, as applied to large amounts of the acid, has 
been described in Folin’s Manual (Folin, 1916), and by Shaffer 
and Hubbard (1916). In this method /3-hydroxybutyric acid was 
oxidized in the presence of 9 to 10 N sulfuric acid, and the amount 
of potassium dichromate was adjusted so that the reaction was 
complete in 15 minutes. 

Continued use of this method showed that there were two 
objections to it; first, the acid was so strong that the tin tubes 
of the ordinary Kjeldahl still were quickly corroded; and second, 
when the technique was extended to very small amounts of 
acetone it was necessary to use a correspondingly decreased 
amount of potassium dichromate. 

In working with conditions in which less sulfuric acid was 
used, a great many different procedures were tried out to find 
which one would give the maximum recovery in a short time. 
It was found impossible to get complete oxidation in 15 minutes, 
and the time was lengthened to half an hour, and potassium 
dichromate was added at intervals instead of by drops. If all 
the reagent was added at once, yields were lower. 

After many trials of different amounts of acid and potassium 
dichromate, and of different methods of adding the dichromate, 
the following was selected: to the /3-hydroxybutyric acid con¬ 
tained in 100 cc. of solution, heated to boiling in a Kjeldahl flask 
attached to a water-cooled condenser, 30 cc. of sulfuric acid 


354 


/3-Hydroxybutyric Acid 


(concentrated sulfuric acid diluted with an equal volume of water) 
and 20 cc. of a potassium dichromate solution, 0.1 to 0.2 per cent, 
were added through a dropping funnel. The burners were regu¬ 
lated so that about 50 cc. distilled in 10 minutes. After 10 minutes 
50 cc. of 0.1 to 0.2 per cent potassium dichromate were added 
and the distillation was continued; 10 minutes later 50 cc. more 
of 0.1 to 0.2 per cent potassium dichromate were added and distil¬ 
lation was continued another 10 minutes. The boiling was not 
interrupted while the additions were made. The total distillate 


TABLE i. 

Determination of Pure Solutions of P-Hydroxybutyric Acid. 
Oxidized for \ hour as described (results are expressed in terms of acetone). 


Present. 

Found. 

Percentage. 

mg. 

mg. 

Tper cent 

0.1202 

0.1036 

86 

0.1202 

0.1050 

88 

0.698 

0.609 

87 

0.698 

0.605 

87 

0.698 

0.610 

87 

1.396 

1.215 

87 

1.396 

1.215 

87 

2.792 

2.405 

86 

2.792 

2.395 

86 

6.01 

5.05 

84 

6.98 

5.90 

84.5 

12.02 

10.25 

85 

24.04 

20.90 

87 

39.53* 

38.2 

86 


* Carried out by oxidizing for hours. 


was collected in a second Kjeldahl flask, and redistilled from 
sodium peroxide for 10 minutes into an Erlenmeyer flask. In 
both distillations a little water was present in the receiving flask, 
and the delivery tube dipped below the surface. Acetone was 
determined in the contents of the Erlenmeyer flask by the method 
described in an earlier paper (Hubbard, 1920). There was a 
blank amounting to 0.01 mg. of acetone. 

Table I shows the results obtained by the method described. 
In these experiments pure solutions of calcium zinc /3-hydroxy- 
butyrate were weighed out, and aliquots were used for each 









R. S. Hubbard 


355 


determination. The “amount present” is expressed as milli¬ 
grams of acetone as calculated from the amount of this salt 
present, and the “amount found” represents the results of the 
titration corrected for the blank on the reagents. The recovery 
ranged from 84 to 88 per cent, and is approximately the same 
given by other methods in which sulfuric acid and potassium 
dichromate are used. The average of the figures is 86 per cent. 
Table I shows that the method as described is applicable for 
amounts of /3-hydroxybutyric acid varying from 0.1 to 25 mg. 

SUMMARY. 

A method is described for the determination of /3-hydroxy¬ 
butyric acid when that compound is present in widely varying 
amounts. The oxidation requires only half an hour, and the 
final determination is by iodometric titration. 

BIBLIOGRAPHY. 

Bergell, P., Z. physiol. Chem., 1901, xxxiii, 310. 

Black, O. F., J. Biol Chem., 1908-09, v, 207. 

Darmstaedter, E., Z. physiol. Chem., 1903, xxxvii, 355. 

Embden, F., and Schmitz, E., in Abderhalden, E., Handbuch der bio- 
chemischen Arbeitsmethoden, Berlin, 1910, iii, 934. 

Folin, O., Laboratory manual of biological chemistry, New York and 
London, 1916. 

Folin, O., and Denis, W., J. Biol. Chem., 1914, xviii, 263. 

Geelmuyden, H. C., Lahf. Forth. Ips., Hammarsten Memorial Number, 11. 
Hubbard, R. S., J. Biol. Chem., 1920, xliii, 43. 

Hurtley, W. H., Quart. J. Med., 1915-16, ix, 301. 

Kennaway, E. L., Biochem. J., 1914, viii, 230. 

Kulz, E., Arch. Biol., 1884, xx, 165. 

Marriott, W. McK., J. Biol. Chem., 1913-14, xvi, 281. 

Magnus-Levy, A., Arch. exp. Path. u. Pharmacol., 1901, xlv, 389. 
Minkowski, O., Arch. exp. Path. u. Pharmacol., 1884, xviii, 35. 

Ohlsson, E., Biochem. Z., 1916, lxxvii, 232. 

Pribram, B. O., Z. exp. Path. u. Therap ., 1911-12, x, 279. 

Shaffer, P. A., J. Biol. Chem., 1908-09, v, 214. 

Shaffer, P. A., and Hubbard, R. S., J. Biol. Chem., 1916, xxiv, p. xxvii. 
Shaffer, P. A., and Marriott, W. McK., J. Biol. Chem., 1913-14, xvi, 265. 
Shindo, S., Inaugural dissertation, Munich, 1907. 

Stadelmann, E., Arch. exp. Path. u. Pharmacol., 1883, xvii, 419. 

Van Slyke, D. D., /. Biol. Chem., 1917, xxxii, 455. 

Wolpe, H., Arch. exp. Path. u. Pharmacol., 1886, xxi, 138. 



Reprinted from The Journal of Biological Chemistry, 
Vol. xlix, No. 2, December, 1921 


DETERMINATION OF THE ACETONE BODIES IN 
URINE.* 

By ROGER S. HUBBARD. 

(From the Laboratory of Biological Chemistry, Washington University 

School of Medicine, Saint Louis, and the Laboratories of The Clifton 
Springs Sanitarium, Clifton Springs, New York.) 

(Received for publication, October 4, 1921.) 

The determination of the acetone bodies in urine has been the 
subject of much investigation. Most of the methods for the 
determination of acetone described previously (Hubbard, 1920) 
and for the determination of /3-hydroxybutyric acid described in 
the preceding note (Hubbard, 1921) have been used to determine 
the acetone bodies in urine, and, in many instances, were pri¬ 
marily developed for that purpose. Besides the references given 
in these two papers, other articles may be found in the bibliog¬ 
raphies in papers by Shaffer (1908-09), Shaffer and Marriott 
(1913-14), Hurtley (1915-16), Van Slyke (1917), and Engfeldt 
(1920). 

The present article contains the description of a method 
which has been found convenient for the determination of acetone 
from preformed acetone plus acetoacetic acid and from /3-hydroxy¬ 
butyric acid on the same sample of urine, even when they are 
present in very small amounts. 

Preliminary Treatment. 

In analyzing urine for the acetone bodies, particularly for 
/3-hydroxybutyric acid, by any method, there are various inter¬ 
fering substances which must be removed. Shaffer (1908-09) 
removed these in two ways: first, by a preliminary precipitation; 
and second, by redistillation to remove compounds other than 

* The work reported in this paper formed a part of a thesis presented 
in partial fulfillment of the requirements for the degree of Doctor of Philoso¬ 
phy at Washington University, Saint Louis, in June, 1921. 

357 


358 


Acetone Bodies in Urine 


acetone which react with alkaline iodine solutions. As a pre¬ 
liminary treatment before analysis he precipitated sugar and other 
interfering substances from urine with basic lead acetate and an 
excess of ammonium hydroxide. When this method was tried 
it was found that the filtrate often contained lead which was pre¬ 
cipitated by subsequent treatment with sulfuric acid. Following 
a suggestion of Plimmer and Skelton (1914), sodium hydroxide 
was substituted for ammonium hydroxide, and it was found that 
glucose could be removed in concentrations up to about 5 per 
cent, and that lead could be completely precipitated at the same 
time, if the quantity of alkali was adjusted so that it was approxi¬ 
mately equivalent to the lead present. During the study reported 
here a paper by Van Slyke (1917) appeared in which the use of 
copper sulfate followed by calcium hydroxide was recommended 
as a preliminary treatment before analysis. This treatment 
removed not only sugar, but other interfering compounds as well 
and was found necessary even when normal urines were analyzed 
by the technique described by him. An older method for remov¬ 
ing sugar from urine was described by Salkowski (1879), in which 
copper sulfate and sodium hydroxide were used. 

The following method was found to give almost complete removal 
of glucose and other easily oxidized compounds. 10 cc. of urine 
were measured into a 250 cc. graduate shaking cylinder, and 
diluted to 100 or 150 cc. 10 cc. of Goulard’s extract 1 and 10 cc. 
of 20 per cent copper sulfate were added, followed by sodium 
hydroxide in not too great excess. (Usually 10 cc. of 2 n concen¬ 
tration were found to be the correct amount for the purpose.) 
This solution was diluted to 250 cc. and filtered after standing 
about half an hour. This combination of the lead and copper 
precipitation methods appeared, in some cases at least, to remove 
interfering (easily oxidizable) compounds more completely than 
did either procedure when used alone. 

There is a relationship between the amount of glucose present 
in urine, and the amount of sodium hydroxide necessary to insure 
its removal by this technique. If 10 cc. of the Goulard’s extract 
and 10 cc. of 20 per cent copper sulfate solution are added to 10 
cc. of normal urine diluted with 100 cc. of distilled water, 5 cc. 
of twice normal sodium hydroxide will not precipitate all the lead 

1 Pb 2 0(CH 3 ' COO) 2 , 290 gm. to 1,000 gm. of solution, prepared accord¬ 
ing to U. S. P., 1916, ix, 249. 


R. S. Hubbard 


359 


from the solution. Under the same conditions, 15 cc. will dis¬ 
solve a part of the lead, while if 7 or 12 cc. are used no lead will 
be found after filtering. If 10 per cent of glucose is present in 
the urine, 15 cc. of 2 n sodium hydroxide will not cause the appear¬ 
ance of lead in the filtrate. 10 cc. of the alkali described above 


TABLE i. 

Precipitation of Urine. 


Glucose. 

NaOH 

Found after precipitation. 

Glucose. 

Lead. 

Copper. 

per cent 

cc. 




0.0 

5 


Trace. 

0 

0.0 

7 


0 

0 

0.0 

12 


0 

0 

0.0 

15 


Trace. 

0 

0.0 

20 

• 

+ 

0 

1.5 

12 

0 

0 

0 

1.5 

15 

0 

Trace. 

0 

2.5 

7 

Very faint trace. 

0 

0 

5 

7 

Trace. 

0 

0 

5 

10 

0 

0 

0 

7.5 

10 

Trace. 

0 

0 

10 

10 

u 

0 

0 

10 

12 

Faint trace. 

0 

0 

10 

15 

0 

0 

0 

15 

15 

Faint trace. 

0 

0 

20 

12 

+ 

0 

0 

20 

15 

Trace. 

Trace. 

Trace. 

20 

20 

+ 

+ 

+ 


Glucose was added to normal urine to give the percentage listed. 10 cc. 
of each were measured into 250 cc. shaking cylinders and diluted to about 
100 cc. 10 cc. of Goulard’s reagent and 10 cc. of 20 per cent copper sulfate 
followed by different amounts of 2n sodium hydroxide were added, and the 
solution was diluted to the mark and filtered at once. The filtrate was 
tested for glucose with Benedict’s solution, for lead with an excess of 
sulfuric acid, and for copper with ammonia. 

will remove glucose up to a concentration of about 5 per cent, 
while larger amounts—15 cc. of 2 n —must be used if the concen¬ 
tration is 10 per cent. Higher concentrations than this were not 
removed by the treatment, and urines containing more than 10 
per cent of glucose must be correspondingly diluted before 
treatment. The facts discussed above are shown in Table I. 














360 


Acetone Bodies in Urine 


For purifying acetone distilled from acetone bodies present 
in urine Shaffer (1908-09) used different methods for the different 
fractions which he determined. He added sodium hydroxide to 
the distillate containing acetone from acetoacetic acid, and redis¬ 
tilled before titrating to remove volatile acids. To the fraction 
corresponding to /3-hydroxybutyric acid he added hydrogen perox¬ 
ide as well as alkali before redistilling to oxidize acetaldeltyde 
and related compounds to the corresponding acids. Folin and 
Denis (1914) used sodium peroxide instead of hydrogen peroxide 
for this purpose. These methods are satisfactory for urines in 
which the acetone bodies are increased to any considerable extent, 
but for normal urines further treatment for the removal of inter¬ 
fering compounds was found to be necessary if the final deter¬ 
mination was to be carried out with dilute alkaline iodine solutions. 
This further purification was accomplished by redistilling first 
from a solution of acid plus potassium permanganate, and then 
by distilling again from sodium peroxide. 

The following directions describe the method used for the 
analysis of the filtrate from the copper and lead precipitation for 
acetone plus acetoacetic acid and for /3-hydroxybutyric acid. 

Determination of Acetone Plus Acetoacetic Acid. 

Measure 150 cc. of the filtrate from urine precipitated as de¬ 
scribed into a 300 cc. Kjeldahl flask, add 10 cc. of sulfuric acid 
(1 part concentrated acid diluted with 1 part water), insert 
a two-holed rubber stopper, with a dropping funnel in one hole and 
a bent distilling tube in the other, connect with a condenser, and 
distil at such a rate that about 50 cc. of distillate come over in 
10 minutes. Collect the distillate in a 500 cc. flask containing a 
little water with the end of the delivery tube below the surface, 
as it should be in all cases in which acetone solutions are distilled, 
and make the distillate up to a volume of about 150 cc. Add to 
the contents of this receiving flask 5 cc. of strong sulfuric acid 
(1 part concentrated acid plus 1 part water), 0.2 gm. of potas¬ 
sium permanganate, and distil, collecting the distillate in a second 
500 cc. flask; continue distillation 10 minutes or more, obtaining 
a final volume of about 100 cc. and taking care that none of the 
permanganate solution boils over. Add to the contents of the 


R. S. Hubbard 


361 


second distilling flask about 0.5 gm. of sodium peroxide, and 
distil 10 minutes into an Erlenmeyer flask containing a little 
water, collecting 50 to 100 cc. If care is not taken at the start, 
the solution will foam over. Cork stoppers should be used for 
this distillation. This technique insures maximum oxidation of 
interfering compounds and does not oxidize acetone (Hubbard, 
1920). When more than mere traces of acetone are present, that 
is, when the urine gives a distinctly positive test with ferric chloride 
or with sodium nitroprusside and alkali, the purification by the 
successive redistillations is unnecessary; for these urines the single 
redistillation from alkali as recommended by Shaffer (1908-09) is 
most satisfactory (see Table II). This technique is to be pre¬ 
ferred under these conditions, not only because it consumes less 
time and takes less apparatus, but also because it reduces chances 
of loss through the vaporization of acetone. 

Determine acetone in the final distillate as follows (Hubbard, 
1920): add 10 to 25 cc. of a solution of iodine in potassium iodide 
of such a strength that 1 cc. is equivalent to 1, 0.1, or 0.2 mg. 
of acetone (the concentration of the iodine to be used is indicated 
by preliminary qualitative tests on the urine); add 2 cc. of a con¬ 
centrated solution of alkali (200 gm. of electrolytic sodium hydrox¬ 
ide dissolved in 300 cc. of distilled water), mix thoroughly, and 
allow to stand for 10 minutes or more; acidify with sulfuric acid, 
and titrate after about 5 minutes with sodium thiosulfate of a 
concentration equivalent to that of the iodine used; add a small 
amount of starch before the end-point is reached to serve as 
indicator. Control titrations of the thiosulfate against iodine 
treated successively with alkali and acid should be run daily, 
as the alkali uses up some of the iodine, and the strength of the 
latter reagent varies somewhat from day to day. 

The difference between this control titration and the titration 
found after distillation measures the acetone present in the sample 
taken, equivalent to 6 cc. of urine. In cases where there is very 
little acetone present it is sometimes necessary to correct for a 
blank given by the reagents after distillation. The question of 
this correction is discussed later. 

The stock iodine solution is prepared by weighing out 13.13 gm. of 
iodine, dissolving with the help of 25 gm. of potassium iodide, and diluting 
to 1 liter. Dilute solutions are prepared from this by diluting with 2.5 per 


362 


Acetone Bodies in Urine 


cent potassium iodide to the desired iodine concentration. These dilute 
solutions change their strength slowly. 

The stock thiosulfate solution of an equivalent strength is made by dis¬ 
solving 25.65 gm. of the pure salt in distilled water. This is standardized 
after 24 hours against an equivalent solution of potassium biiodate con¬ 
taining 3.362 gm. per liter, and protected from the action of carbon dioxide 
with soda lime; so protected, the solution will keep its strength unchanged 
for months. The dilute solutions are not as stable, and are prepared from 
this stock solution daily. 

In Table II are given the values obtained for acetone from 
acetone plus acetoacetic acid as found after distilling a sample 
of normal urine, and of the filtrate from the copper and lead treat¬ 
ment of the same urine, from various oxidizing reagents. The 


TABLE II. 

Effect of Successive Distillation from Different Reagents on Urine Acetone. 


Distilled successively from reagents given. 

Straight 
urine in 

Precipitated 
urine in 


6 cc. 

100 cc. 

6 cc. 

100 cc. 

h 2 so 4 . 

mg. 

0.76 

mg. 

12.7 

mg. 

0.6 

mg. 

10.0 

H 2 S0 4 ; NaOH. 

0.11 

1.8 

0.10 

1.7 

H 2 S0 4 ; Na 2 0 2 . 

0.17 

2.8 

0.16 

2.7 

H 2 S0 4 ; H 2 S0 4 + KMn0 4 .. 

0.14 

2.3 

0.11 

1.8 

H 2 S0 4 ; H 2 S0 4 T KMn0 4 ; Na 2 0 2 . 

0.05 

0.8 

0.03 

0.5 

H 2 S0 4 ; H 2 S0 4 + KMn0 4 ; Na 2 0 2 . 

0.0462* 

0 .8* 

0.0496* 

0 .8* 


* Carried out on a different sample of urine and titrated with a weaker 
solution of thiosulfate. All results are expressed in terms of acetone. 


repeated distillation is shown to be necessary when the amount 
of acetone is very small, but not necessary when it is increased 
as, in the latter case, the very small difference lies' within the 
limits of experimental error. The table also shows agreement 
between figures on untreated urine and on filtrates from the pre¬ 
cipitation with copper and lead when the distillate is purified by 
successive redistillations. 

Determination of (3-Hydroxybutyric Acid. 

To determine the /3-hydroxybutyric acid in the urine, treat the 
contents of the first distilling flask (urine filtrate plus sulfuric acid) 
by the technique described in the preceding note (Hubbard, 1921). 

















R. S. Hubbard 


363 


To the boiling solution add, through the dropping funnel, 20 cc. 
of the strong sulfuric acid (1 part concentrated acid plus 1 
part water), 30 cc. of 0.1 to 0.2 per cent potassium dichromate, 
and continue the determination as described for solutions of pure 
/3-hydroxybutyrie acid. Redistil the acetone obtained as in the 
case of the first fraction from sulfuric acid plus potassium perman¬ 
ganate and from sodium peroxide, and carry out the determination 
on the final distillate in the manner described for the fraction 
from preformed acetone plus acetoacetic acid. This determina- 


TABLE III. 

Duplicates on Urines. 


Redistillations. 

Urine 

No. 

Acetone + acetoacetic 
acid. 

/3-Hydroxybutyric 

acid. 

Sample. 

Per 100 cc. 

Sample. 

Per 100 cc. 



mg. 

mg. 

mg. 

mg. 

From Na 2 0 2 *. 

1 

0.067 

0.7 

0.180 

1.8 


1 

0.080 

0.8 

0.290 

2.9 


1 

0.077 

0.8 

0.200 

2.0 


1 

0.035 

0.35 

0.183 

1.8 


1 

0.045 

0.45 



Prom H 2 S0 4 + KMn0 4 f 






and from Na 2 0 2 . 

2 

0.078 

1.3 

0.130 

2.2 


2 

0.084 

1.4 

0.130 

2.2 


2 

0.072 

1.2 

0.110 

1.8 


2 

0.0961 

1 .6J 

0.1101 

1 .8t 


2 

0.0781 

13J 




* Samples of 10 cc. each used for these determinations, 
f Samples of 6 cc. each used for these determinations, 
t Glucose added to the urine to give a concentration of 5 per cent. 


tion gives the acetone formed from the oxidation of the /3-hydroxy- 
butyric acid present. A correction of 15 per cent must be added 
to the result to make up for the incomplete recovery of acetone. 
If much acetone is present the redistillation from sulfuric acid 
plus potassium permanganate may be omitted. 

Table III shows the agreement between duplicates obtained by 
this technique on both fractions of acetone from normal urine, 
as contrasted with the agreement when the redistillation from 
acid plus potassium permanganate was omitted. 













364 


Acetone Bodies in Urine 


Recovery of Substances Added to Urine. 


Table IV shows the recovery of acetone, acetoacetic acid, and 
/3-hydroxybutyric acid by this method. The acetone used was puri¬ 
fied by repeated redistillation until the boiling point was constant. 
The acetoacetic acid was prepared by hydrolyzing acetoacetic 
ethyl ester with sodium hydroxide, aerating to remove acetone, 
and acidifying. The product was then analyzed by distilling 
from sulfuric acid, and redistilling from sulfuric acid plus potas¬ 
sium permanganate and from sodium peroxide to remove alcohol, 
and titrating the acetone formed by the usual Messinger method. 
The /3-hydroxybutyric acid was prepared from calcium zinc 
/3-hydroxybutyrate which was shown, by its optical activity, to be 
99 per cent pure. A solution of this salt was acidified, set in plaster, 
and extracted with ether for about 10 hours. The ether was 
distilled off, and the /3-hydroxybutyric acid taken up wfith water. 
The solution was boiled with a little bone-black, filtered, made 
up to 100 cc. with distilled water, and read in a polariscope. 
The reading in a 2.2 dm. tube was 0.865. This corresponds to a 
concentration of 1.630 gm. in 100 cc. (two determinations). 


0.865 

2.2 X 24.12 


1.630 


Two dilute solutions were prepared from this by diluting 1 to 10. 
A 5 cc. portion of each was analyzed by the technique described in 
the preceding note (Hubbard, 1921), and it was found that the 
acetone recovered was 85 per cent of the theoretical amount, the 
usual percentage recovered by this oxidation. The recovery of 
these substances when added to normal urine was satisfactory 
(Table IV). 

Blanks on Reagents. 

In all determinations in which distillation precedes the final 
analysis with dilute iodine solutions there is a blank. Its value 
is small when measured in terms of milligrams of acetone, but 
may amount to a high percentage of the total determinations 
when very small quantities of acetone are present. A large num¬ 
ber of experiments were carried out to determine the source of 
this blank, and to find out conditions which should reduce it to 



R. S. Hubbard 


365 


a minimum. Many of the precautions which were used to obtain 
very low blanks have been since found described by Widmark 
(1919) in his paper on the determination of acetone in blood. 

TABLE IV. 


Recovery of Substances Added to Urine. 


Concentra¬ 

tion 

prepared per 
100 cc. 

Taken. 

Found. 

Calculated 
per 10 cc. 

Added per 
10 cc. 

Per cent. 

Filtrate. 

Urine. 

Acetone. 

mg. 

cc. 

cc. 

mg. 

mg. 

mg. 


2.6 

150 

6 

0.088 

0.146 

0.144 

101 

5.8 

150 

6 

0.178 

0.296 

0.289 

102 

14.8 

150 

6 

0.424 

0.707 

0.739 

96 

30.4 

150 

6 

1.00 

1.67 

1.52 

110 

60.0 

150 

6 

1.88 

3.13 

3.00 

104 

149.0 

150 

6 

4.32 

7.20 

7.45 

97 

600 

150 

6 

17.5 

29.2 

29.98 

97.5 


Acetoacetic acid. 


2.6 

150 

6 

0.0656 

0.109 

0.108 

100 

25.8 

150 

6 

0.834 

1.39 

1.29 

108 

60.6 

150 

6 

2.07 

3.46 

3.32 

104 

117 

150 

6 

3.82 

6.37 

6.41 

100 

132 

150 

6 

3.87 . 

6.47 

6.64 

98 

320 

150 

6 

9.55 

15.92 

16.01 

100 

638 

150 

6 

19.67 

32.70 

31.97 

102.5 

684 

150 

6 

18.23 

30.4 

34.2 

90 


0 -Hydroxybutyric acid. 


4.5 

150 

6 

0.230 

0.382 

0.454 

85 

9.1 

150 

6 

0.470 

0.785 

0.909 

86 

18.2 

150 

6 

0.975 

1.62 

1.82 

89 

45.45 

150 

6 

2.27 

3.90 

4.545 

84 

90.9 

150 

6 

4.69 

7.81 

9.09 

86 

181.8 

150 

6 

9.54 

15.9 

18.2 

87.5 

454.5 

150 

6 

22.24 

37.15 

45.45 

82 


All results and figures are given in terms of acetone. 


One source of the acetone value found in blank determinations 
seems to be the presence of a very small amount of some impurity 
in the reagents, possibly in the lead subacetate used in precipi- 




































366 


Acetone Bodies in Urine 


tating the urine, but this forms only a small percentage of the 
total value. The larger part of the blank is present when water 
alone is distilled successively from the different reagents, and 
seems to come largely from the last distillation from sodium 
peroxide. Cork stoppers must be used to connect the distilling 
flask with the condenser, unless, as suggested by Widmark, an 
all glass still is available. It is necessary to boil water through this 
still before each determination, and it was found best to boil a 
solution containing the same amount of sodium peroxide in the 
flasks before they were used for this final distillation. This pro¬ 
cedure again resembles that recommended by Widmark. The 
still and flask used in the distillation with acid and potassium 


permanganate were 

similarly boiled out 

TABLE V. 

each day to remove 

Date. 

Acetone + acetoacetic acid. 

/3-Hydroxybutyric acid. 

im 

mg. 

mg. 

Sept. 11 

0.0125 

0.0185 

“ 11 

0.0125 

0.0165 

“ 11 

0.0138 

0.0131 

“ 12 

0.0147 

0.0190 

“ 12 

0.0262 

0.0189 

“ 12 

0.0151 

0.0135 - 


All results are expressed in terms of the acetone equivalent of the blank. 
These blanks were obtained by redistilling the first distillate from H2SO4 + 
KM11O4 and from Na 2 C >2 successively. 


grease which might yield, on oxidation, substances reacting with 
alkaline iodine solutions. Another source of error which should 
be avoided is the presence in the air of the laboratory of ammonia, 
formaldehyde, reducing gases, and other fumes which react with 
alkaline iodine solutions. In working with dilute reagents such 
as are used compounds of this nature may cause serious complica¬ 
tions. If, however, the analyses are carried out on urines in 
which the acetone content is only slightly increased, these pre¬ 
cautions may be omitted. In Table V a number of determina¬ 
tions of blanks on urine reagents treated as in the determination 
are given; these blanks show good agreement with each other. 
It is noticeable that the values of the blanks are so small that they 
are of importance only when there is very little acetone present. 









R. S. Hubbard 


367 


In Table YI the results obtained by titrating the distillates 
finally obtained from normal urines are compared with results 
obtained by the use of the reagent described by Scott-Wilson 
(1911). Acetone gives a turbidity with this reagent and the 
reaction is a very delicate one. The turbidity obtained from the 
acetone was matched against that produced by a known amount 
of acetone freshly distilled into the reagent. In some cases the 
turbidities were matched in Nessler tubes; determinations could 
be made to an accuracy of about 0.005 mg. by this technique. 
In other cases the solutions were read in the nephelometer 
(Marriott, 1913-14), and when the amounts of acetone were 
comparatively large, the colorimeter as described by Folin and 


TABLE vi. 

Comparison of Urine Acetone by Iodine and by Scott-Wilson Reagent. 



Acetone + acetoacetic acid. 

/3-Hydroxybutyric acid. 

No. 

Scott-Wilson reagent. 

Iodine titration. 

Scott-Wilson reagent. 

Iodine titration. 


6 cc. 

Concen¬ 
tration 
per 100 cc. 

6 cc. 

Concen¬ 
tration 
per 100 cc. 

6 cc. 

Concen¬ 
tration 
per 100 cc. 

6 cc. 

Concen¬ 
tration 
per 100 cc. 


mg. 

mg. 

mg. 

mg. 

mg. 

mg. 

mg. 

mg. 

1 

0.033 

0.55 

0.033 

0.55 

0.090 

1.5 

0.113 

1.9 

2 

0.025 

0.4 

0.023 

0.4 

0.083 

1.4 

0.075 

1.25 

3 

0.027 

0.45 

0.032 

0.6 

0.130 

2.2 

0.120 

2.0 


Three normal urines used. All results are expressed in terms of acetone. 


Denis (1914) was used to measure the turbidity. Good agree¬ 
ment was found by the two methods, and it seems certain that the 
results must represent acetone. Whether the source of that ace¬ 
tone in the determinations of /3-hydroxybutyric acid was actually 
that acid present in very small amounts in the normal urines 
analyzed is a question which the results do not establish. 

Table VII contains results obtained by the use of the method 
described on a number of urines. The cases included range from 
normal subjects to diabetics showing a moderately advanced degree 
of acetonuria. A few facts are noticeable. First, the values 
found for the normals are very low, in the vicinity of about 2 mg. 
per 100 cc. of urine, for acetone from all acetone bodies. These 
results correspond with the lowest values included in the literature. 





















368 


Acetone Bodies in Urine 


TABLE VII. 

Urine Determinations. 


Urine 

No. 

Sex. 

Date. 

Preformed 
acetone + aceto- 
acetic acid. 

!/3-H ydroxybuty- 
ric acid. 





mg. per 
100 rc. 

' gm. per 
2If. hrs. 

mg. pei 
100 cc. 

• gm. per 
2If hrs. 

1 

Male. 



1.3 


2.4 


2 

CC 



0.5 

0.007 

2.5 

0.030 

3 

CC 



0.7 

0.009 

1.6 

0.021 

4 

Female. 



0.4 

0.001 

1.4 

0.010 

5 

Male. 



1.6 

0.003 

2.3 

0.009 

6 

CC 



0.8 

0.002 

1.6 

0.003 

7 

Female. 

Mar. 13, 1921 

1.9 

0.016 

1.2 

0.010 

8 

u 

Feb. 

17, 1921 

0.2 

0.004 

0.5 

0.009 

9 

CC 

Mar. 11, 1921 

3.2 

0.026 

2.0 

0.016 

10 

cc 

Nov. 10, 1919 

1.1 

0.006 

6.1 

0.040 

11 

cc 

Dec. 

7, 1920 

19.6 

0.192 

19.4 

0.190 

12 

Male. 

Aug. 15, 1919 

0.4 


1.3 




CC 

19, 1919 

2.1 

0.024 

1.5 

0.017 

13 

Male. 

CC 

17, 1919 

0.5 

0.009 

1.2 

0.020 



cc 

19, 1919 

1.5 

0.025 

1.7 

0.028 

14 

Female. 

cc 

17, 1919 

11.2 

0.182 

15.0 

0.244 



cc 

18, 1919 

9.2 

0.156 

17.8 

0.302 



cc 

19, 1919 

11.2 

0.063 

22.9 

0.128 

15 

Female. 

cc 

18, 1919 

15.9 

0.312 

20.5 

0.403 


. 

cc 

19, 1919 

7.2 

0.163 

4.9 

0.110 



cc 

20, 1919 

3.3 

0.092 

4.0 

0.011 

16 

Female. 

cc 

18, 1919 

1.6 

0.021 

4.3 

0.055 



cc 

19, 1919 

1.3 

0 017 

3.3 

0.043 



cc 

20, 1919 

2.5 

0.024 

5.3 

0.052 

17 

Male. 

cc 

15, 1919 

6.4 

0.204 

6.3 

0.203 



cc 

17, 1919 

5.1 


8.8 




cc 

18, 1919 



6.24 

0.176 



cc 

19, 1919 

3.0 

0.101 

1.7 

0.057 

18 

Female. 

cc 

15, 1919 

0.4 

0.007 

2.1 

0.041 



cc 

17, 1919 

1.3 

0.058 

3.4 

0.058 



cc 

18, 1919 

4.4 

0.085 

4.3 

0.079 



cc 

19, 1919 

1.7 

0.032 

2.5 

0.048 


Remarks. 


Normal mixed. 
“ case 1. 

cc cc 2 

“ child. 

cc cc 

cc cc 

Arthritic. 

“ case 

cc cc 

Overweight. 

Diabetic. 

cc 


Diabetic. 


Diabetic. 


Diabetic. 


Diabetic. 


Diabetic. 


Diabetic. 


CO ^ 



























R. S. Hubbard 

TABLE VII— Concluded. 


369 


•o 

•gfc 

p 

Sex. 

Date. 

Preformed 
acetone + aceto¬ 
acetic acid. 

/8-Hydroxybuty- 
ric acid. 

Remarks. 





mg. per 
100 cc. 

gm. per 
U hrs. 

mg. per 
100 cc. 

gm. per 
24 hrs. 


19 

Female. 

Aug. 15, 1919 

1.3 

0.037 

2.2 

0.065 

Diabetic. 



u 

17, 1919 

1.5 

0.052 

2.7 

0.092 




u 

18, 1919 

2.7 

0.041 

3.2 

0.069 




it 

19, 1919 

0.5 

0.012 

1.2 

0.027 


20 

Male. 

Nov. 30, 1920 

24.3 

0.467 

20.8 

0.400 

Diabetic case 5. 



Dec. 

1, 1920 

34.3 

0.686 

54.3 

1.28 




iC 

2, 1920 

49.4 

0.673 

137 

1.86 




U 

3, 1920 

56.1 

0.822 

137 

2.00 




u 

5, 1920 

61.0 

1.12 

150 

2.77 




u 

7, 1920 

75.5 

1.32 

199 

3.48 




u 

9, 1920 

53.0 

1.08 

107 

2.49 



All results are expressed in terms of acetone. 


Second, there is, in most normal cases more acetone from 
0-hydroxybutyric acid than from acetone plus acetoacetic acid. In 
cases in which the excretion of the acetone bodies is slightly in¬ 
creased these two fractions are nearly equal, and in some of these 
cases there is an excess of the fraction from preformed acetone 
plus acetoacetic acid over that from /3-hydroxy butyric acid. In 
the cases in which larger amounts of these substances were ex¬ 
creted the fraction from /3-hydroxybutyric acid was again found 
to be the larger, as has been repeatedly found in cases of diabetes 
in which there was a marked degree of acetonuria. These facts 
militate against the theory formerly held that there is any definite 
ratio between the amounts of the acetone bodies formed in the 
body which can be studied from the proportions excreted (see 
also Hurtley, 1915-16). In the two following experiments it is 
shown that these same relationships occur in the same individual 
under conditions which lead to a gradual development of ace¬ 
tonuria. 

In Table VIII 2 are recorded data obtained on a normal subject 
while he was living on a diet the fat content of which was increased. 


2 This experiment was carried out in the metabolic ward of Barnes 
Hospital, Saint Louis, through the courtesy of Dr. William H. Olmstead. 













TABLE VIII. 


370 


Acetone Bodies in Urine 



O 

V 


O 05 

05 

CO 

05 


05 i-i 

© 

© 

Ttl 


& 

• 

CO -H 

CO 

O 

© 

05 

© © 

05 

05 




S 

© o 

o 

05 

© 


CO © 

© 

© 

© 


3 

C3> 











x> . 


© o 

o 

© 

© 

rH 

© © 

© 

© 

© 


>)t3 












rs • H 

o « 

(h ej 

. 

s> ^ 

05 00 

to 

oo 

© 

© 

© 00 



© 


>> 

ft. u 

05 CO 

CO 

CO 

© 

05 

© © 

CO 

CO 

d 


w 


rH 

© 

rH 

CO 






s ^ 





rH 






o 


CO 00 

ss 

to 


CO 

05 

B- 

© 

CO 

TH 



• 

CO 

b- 


© 

© 

05 

05 

05 


o 

s 

o 

CO 


00 

05 tH 

© 

© 

© 

GQ 

3 

o3 

+1 

01 a 

<2b 

© o 

d 

© 

© 

© 

© © 

© 

© 

© 

A 

Tt> 



05 

05 

© 

© 

© CO 

co 

00 


CM 

o 

ft « 

d ^ 

1> 

TtH 

00 

CO 

00 © 

CO 

05 

rH 

<D 

a> 

go 



CO 

© 

© 

05 rH 




ft 

(D 

« 

S'" 










a 












TJ 

£ 


00 00 

CO 

r^ 

© 

"'t 1 

T* © 

© 

u- 

© 

p 


• 

TjH ^ 

B- 

00 

rH 

rH 

© HjH 


© 

© 


w 

s 

t> b- 

1> 


00 

© 

© CO 

© 

© 



s; 


o o 

o 

© 

© 

© 

© © 

© 

© 

© 


& 








© 





s 

© o 

CO 

CO 

© 

© 

Tf CO 


© 

© 


-H 

o> 

05 t- 

CO 


d 

05 

CO © 

© 

© 

© 


o 

Eh 


r— 1 H 

rH 

rH 

rH 

rH 

rH rH 


rH 



6 

G 


to o 

to 

to 

© 

© 

© © 

© 

© 

© 



CO O 

o 

© 

rH 

00 

00 ^ 

© 

rH 

rH 


a 

8 

05 i - 1 

05 

© 

00 

rH 

© © 

CO 

00 

© 


o 












> 


vH 


rH 


rH 

• rH 





OQ 

O 


CO CO 

CO 

rH 

rH 

rH 

oo co 

00 

00 

co 




B- B- 

I- 

rH 

rH 

rH 

'OH tH 
tJH 

-n 

rf 



jo 


to to 

to 

t- 


i> 




c3 

o 


of of 

of 

of 

of 

of 

of of 

of 

of 

of 



HO 

£ 

to to 

to 

to 

© 

© 

© © 

© 

© 

© 


6 









-M 


to to 

to 



t- 

d d 

d 

d 

d 


T3 

>1 

fe 

ft 

rH rH 

rH 




05 05 

05 

05 

05 














o 












rO 


o o 

o 

© 

o 

© 

© © 

© 

© 

© 


o3 

S 

o o 

o 

to 

© 

© 

© © 

© 

© 

© 


o 

05 

rH rH 

rH 




rH rH 

rH 

rH 

rH 



"s 

to to 

to 


rH 


B- I> 



5> 


* 











o; 



05 05 

05 

05 

05 

05 

CO CO 

CO 

co 

CO 

Q 



CO CO 

CO 

00 

CO 

CO 

© © 

© 

© 

© 


Fat. 

ft 











s' 

H rH 

rH 

rH 

rH 

rH 

© © 

© 

© 

© 




o o 

© 

© 

© 

© 

B- B- 


b- 

t- 



Ob 

05 05 

05 

05 

05 

05 

rH rH 

rH 

rH 

rH 



£ 



rH 

rH 

rH 

b- b- 

i> 


i> 



o 

to to 

to 

© 

© 

© 

rH rH 

rH 

r-J 

rH 



8 

rH rH 

rH 

rH 

rH 

rH 

rH rH 

rH 

rH 

rH 


• »H 

ft 























o 

























* 

CO CO 

© 

00 

OC 

CO 

05 05 

05 

05 

05 



s 

05 05 

© 

© 

© 

© 

b- t- 


b- 

r- 



05 














V 











05 CO 


© 

© 


00 © 

© 

rH 

05 




rH rH 

rH 

rH 

rH 

rH 

rH rH 

05 

05 

05 


H> 


• 










c3 

CO 

a. 










Q 


c3 v. 

V, 

V# 



v. ^ 







»—i - 












Results of determinations of the acetone bodies are expressed in terms of acetone. 








































R. S. Hubbard 


371 


The subject of the experiment (the author), was a man 5 feet, 
10| inches in height, weighing 165 pounds, who was doing light 
laboratory work at the time. The amounts of fat eaten were as 
follows: 200 gm. of fat during the first 3 days of the experiment; 
250 gm. during a second period of the same duration; and 175 gm. 
during an after period of 5 days. The carbohydrate was also, 
varied during the different periods as shown. There was a slight 
increase in the acetone excretion during the first period, a marked 
increase during the second period, and a return to practically 
normal values at the end of the experiment. Since the appearance 
of a paper by Shaffer (1921) on the relationship of glucose and 
fat to each other in diets which show acetonuria, the percentage 
of the total calories fed as fat, as protein, and as carbohydrate 
have been calculated, and are given in the table. It is noticeable 
that the first diet taken, which caused a slight increase in the 
excretion of acetone, was markedly higher in actual and available 
carbohydrate than the one which he has described as the border¬ 
line diet for acetonuria; that is, one containing the foods in the 
ratio of 10 per cent in the form of carbohydrate, and 80 per cent 
in the form of fat. The excretion of acetone on this diet was 
increased only very slightly, however, and can, perhaps, be prop¬ 
erly attributed to variations in the mixtures of fat and carbo¬ 
hydrate burned at different times during 24 hours; with diets at 
or on the border-line of producing acetone it is evident that such 
variations may be important in causing slight increases in the 
excretion of acetone. 

The relationship between the two fractions of acetone referred 
to above is shown in this experiment. At the start there was an 
excess of acetone from /3-hydroxybutyric acid over that from ace¬ 
tone plus acetoacetic acid, a condition often, although not invari¬ 
ably, found in normal urine. During the first part of the experi¬ 
ment the fraction from acetone was in excess, but when the diet 
was markedly high in fat, and the total excretion of acetone 
consequently increased, the relationship of the substances was 
the same as that found at the start of the experiment; that is, 
there was an excess of acetone from /3-hydroxybutyric acid similar 
to that usually described in diabetic urines. When the diet became 
more nearly normal, and the excretion of acetone began to de¬ 
crease, there was again a period in which there was more acetone 


0 


372 Acetone Bodies in Urine 

from preformed acetone plus acetoacetic acid than there was 
from jS-hydroxybutyric acid. 

The total nitrogen (determined by the Kjeldahl method) 
shows a slight negative balance during the first part of the experi¬ 
ment, but this negative balance was not sufficiently pronounced 
to allow conclusions to be drawn from the data. The ammonia 
nitrogen excretion was increased, and roughly paralleled the 
increased acetone excretion. The ammonia was determined by 
the method of Folin and Macallam (1912). 

Table IX, which contains results from another normal subject 
observed during a short fast, shows somewhat the same picture 


TABLE IX. 

Acetone Bodies Excreted during a Short Fast. 


Day of fast. 

Before.* 

First. 

Second. 

Third. 

Day 

after. 

Urine volume, cc . 

1,340 

1,120 

1,020 

1,265 

1,270 

Acetone + acetoacetic acid, mg. per 
100 cc . 

0.7 

0.4 

11.0 

14.0 

4.5 

Acetone + acetoacetic acid, gm. per 
24 hrs . 

0.009 

0.0045 

0.112 

0.177 

0.057 

/3-Hydroxybutyrie acid, mg. per 100 cc... 

1.6 

1.4 

4.0 

9.0 

3.8 

/3-Hydroxybutyric acid, gm. per 24 hrs... 

0.021 

0.016 

0.041 

0.114 

0.043 


* This sample of urine was obtained from the subject several days 
before the experiment was commenced. 

All results are expressed in terms of acetone. 


although here the experiment was not carried far enough to show 
the second crossing of the curves which is known to take place in 
cases of prolonged fasting. This case resembles, in the general 
course of the relative concentrations of the fractions, the cases 
reported by Folin and Denis (1915). 

Many cases of diabetes studied during the development and 
clearing up of acetonuria have shown similar phenomena, and it 
can be found also in some of the results in the literature (Hurtlev, 
1915-16). 

As it is generally accepted that acetoacetic acid is the immediate 
source of preformed acetone (Folin, 1907; Widmark, 1920) these 
two compounds can be considered together, and the acetone of 


















R. S. Hubbard 


373 


the breath added to give a total measure of the acetoacetic acid 
excreted. From the data presented, there would appear to be a 
marked increase in the acetoacetic acid before there is an increase 
in /3-hydroxybutyric acid; if this fact, shown by a study of the 
excretion of the “ acetone bodies, ” is a true measure of the relative 
formation of the two compounds in the body, it would seem to 
indicate that the substance which finally fails to be completely 
burned under the conditions studied is acetoacetic acid rather 
than /3-hydroxybutyric acid. On the other hand, it is possible 
that the excretion of acetoacetic acid occurs at a lower concentra¬ 
tion of the substance in the blood than does the excretion of 
/3-hydroxybutyric acid. This is known to be the case for acetone 
itself as compared with acetoacetic acid (Widmark, 1920), and it 
is not unreasonable to assume that there may be a similar relation¬ 
ship between the “kidney thresholds” for the other two acetone 
bodies. In one case a sample of blood was obtained from a patient 
during the development of acetonuria when there was a higher 
concentration of acetone from acetone plus acetoacetic acid in the 
urine than there was from /3-hydroxybutyric acid, and in the 
blood these two fractions were found to stand in the opposite 
relationship to each other. For the present, attention can only 
be called to the fact that there is such a difference in the excretion 
of the two fractions when the organism is producing only slightly 
increased amounts of the acetone bodies. 

• CONCLUSION. 

A method has been described for the determination of the 
acetone bodies in normal urine, which gives a good percentage of 
recovery for substances added, and which is particularly applicable 
for the analysis of normal urines. It gives low values for normal 
urines, but not lower than some already included in the literature. 
Duplicates agree well, and determinations when carried out by 
two distinct methods of final analysis show satisfactory agree¬ 
ment. In addition two cases are presented in which the gradual 
development of acetonuria was brought about, and a brief dis¬ 
cussion is given of the relationship between the different acetone 
bodies under such conditions. 

My thanks are due to Dr. Philip A. Shaffer for his advice and 
assistance during the progress of this work. 


374 


Acetone Bodies in Urine 


BIBLIOGRAPHY. 

Engfeldt, N. 0., Beitrage zur Kentnisse der Biochemie der Acetonkor- 
per, Lund, 1920. 

Folin, 0., J. Biol. Chem., 1907, iii, 177. 

Folin, O., and Denis, W., J. Biol. Chem., 1914, xviii, 263. 

Folin, O., and Denis, W., J. Biol. Chem., 1915, xxi, 183. 

Folin, O., and Macallam, A. B., J. Biol. Chem., 1912, xi, 523. 

Hubbard, R. S., J. Biol. Chem., 1920, xliii, 43. 

Hubbard, R. S., J. Biol. Chem., 1921, xlix, 351. 

Hurtley, W. H., Quart. J. Med., 1915-16, ix, 301. 

Marriott, W. McK., J. Biol. Chem., 1913-14, xvi, 281. 

Plimmer, R. H. A., and Skelton, R. F., Biochem. J ., 1914, viii, 64. 
Salkowski, E., Z. physiol. Chem., 1879, iii, 79. 

Scott-Wilson, H., Biochem. J., 1911, xlii, 444. 

Shaffer, P. A., J. Biol. Chem., 1908-09, v, 211. 

Shaffer, P. A., J. Biol. Chem., 1921, xlvii, 449. 

Shaffer, P. A., and Marriott, W. McK., J. Biol. Chem., 1913-14, xvi, 265. 
Van Slyke, D. D., J. Biol. Chem., 1917, xxxii, 455. 

Widmark, E. M. P., Biochem. J., 1919, xiii, 430. 

Widmark, E. M. P., Biochem. J., 1920, xiv, 364. 


Reprinted from The Journal of Biological Chemistry, 
Vol. xlix, No. 2, December, 1921 


DETERMINATION OF THE ACETONE BODIES IN 
BLOOD.* 

By ROGER S. HUBBARD. 

{From the Laboratory of Biological Chemistry, Washington University School 
of Medicine, Saint Louis, and the Laboratories of The Clifton 
Springs Sanitarium, Clifton Springs, New York.) 

(Received for publication, October 4, 1921.) 

Comparatively few methods are available for the determination 
of the acetone bodies in blood. Marriott (1913-14, b) has 
described a method by which the nephelometric technique 
described by him (1913-14, a) could be applied to the determina¬ 
tion of these substances in blood. Scott-Wilson’s reagent was 
used in this determination (Scott-Wilson, 1911). Marriott used 
this method in studying the relationships of the acetone bodies 
to each other (1914, b) and the level of acetone content of the 
blood in acidosis (1914, a). 

Kennaway (1914) has described a method for determining 
acetone bodies in urine, using the Scott-Wilson reagent for the 
final determination. In a later paper Kennaway (1918) has 
applied this technique to the determination of acetone in the 
blood from diabetic patients, but was not able to carry through 
the method successfully on normal bloods. 

Van Slyke and Fitz (1917, 1920) have described the application 
of Van Slyke’s (1917, b) method to the determination of acetone 
bodies in blood. Short (1920) has recently described a modi¬ 
fication of this method applicable to blood from operative cases 
in which ether is used. 

Widmark (1919) has described a method for determining acetone 
from preformed acetone plus acetoacetic acid by the use of a 

* The results reported in this paper formed a part of a thesis presented 
in partial fulfillment of the requirements for the degree of Doctor of Phil¬ 
osophy at Washington University, Saint Louis, in June, 1921. A partial 
preliminary report of the method was given before the American Society 
of Biological Chemists, in December, 1920 (Hubbard and Wright, 1921). 

375 


THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. XLIX, NO. 2 


376 


Acetone Bodies in Blood 


modified Messinger titration method. This method, in common 
with those described by Ljungdahl (1917, 1919) and others, used 
a solution of biiodate as the source of the iodine. Widmark’s 
method does not show the presence of acetone from preformed 
acetone plus acetoacetic acid in normal blood, and, in common 
with most of the other methods, is not applicable for the deter¬ 
mination of /3-hydroxybutyric acid in the same sample in which 
acetone is determined. 

In extending the method for the determination of small amounts 
of .acetone by the Messinger titration (Hubbard, 1920) to the 
determination of the acetone bodies in blood the only difficulty 
found was in choosing a satisfactory method for the precipitation 
of protein and other interfering compounds. Marriott (1913-14, b) 
used colloidal iron for removing protein before determining ace¬ 
tone with Scott-Wilson’s reagent, and by modifying this pro¬ 
cedure it was found possible to remove a large part of the reduc¬ 
ing compounds with the protein, and so make the technique 
available for a final determination with alkaline iodine solutions 
instead of with the reagent used by him. The method used was 
based on experiments with lead and sodium hydroxide described 
in the preceding paper (Hubbard, 1921). 

An amount of blood varying from 1 to 5 cc. was measured into 
a 100 cc. shaking cylinder, and water was added to give a volume 
of between 40 and 50 cc. 50 cc. of colloidal iron were added, and 
the solution was thoroughly shaken. Next, 10 cc. of Goulard’s 
extract were added, and the solution was shaken again. Finally, 
enough sodium hydroxide was added to precipitate the lead, the 
solution again thoroughly mixed, and allowed to stand for about 
1 hour. It was then diluted to the mark and centrifuged in tubes 
covered with rubber caps to prevent loss of acetone. The super¬ 
natant liquid was then poured through filter paper, and an aliquot 
used for the analysis. The filtrate was clear, gave no precipitate 
of lead with sulfuric acid, or of protein with sulfosalicylic acid, 
and contained very little reducing substances when tested with 
alkaline picrate solutions even when the concentration of blood 
sugar was as high as 0.3 per cent. 

The amount of sodium hydroxide that should be used must 
be determined by preliminary experiment for each batch of 
Goulard’s reagent as the lead content of this reagent varies some- 


R. S. Hubbard 


377 


what. The proper amount to use is the smallest amount of sodium 
hydroxide which will precipitate the lead from solution. The 
filtrate from the precipitation of lead with sodium hydroxide 
should react alkaline to litmus, but only faintly alkaline to phenol- 
phthalein. 50 cc. when titrated with 0.01 n acid will require only 
about 2 cc. to render it neutral to phenolphthalein. 10 cc. of the 
reagent used in this work required 5.9 cc. of 2 n sodium hydroxide 
to give the required end-point. Electrolytic sodium hydroxide 
should be used in preparing this reagent. 

An aliquot of the filtrate was used for the determination of the 
acetone bodies. If only 5 cc. of blood were available for the 
determination, the aliquot taken was usually 50 cc. When more 
blood was available two or three separate precipitations were 
carried out as described, and the filtrates combined. In all cases 
the solution was diluted to a final volume of 150 cc. for the deter¬ 
mination. The accuracy of the determination is, of course, 
greatly increased when more blood is available. This analysis was 
carried out in the same way that was described for urine, except 
that it was found that a distillation of the distillate first obtained 
from an excess of sodium hydroxide before treatment with sulfuric 
acid plus potassium permanganate gave more consistent results. 
This treatment appeared to remove some volatile acid which, on 
oxidation with acid and potassium permanganate gave a compound 
which reacted with alkaline iodine solutions. Subsequent dis¬ 
tillations from sulfuric acid plus potassium permanganate and 
from sodium peroxide were carried out as described in the pre¬ 
ceding paper. For the final determination it is advisable, in 
most cases, to use 10 cc. of an iodine solution, 1 cc. of which is 
equivalent to 0.02 mg. of acetone, and titrate with a solution of 
sodium thiosulfate which is one-half as concentrated. In this 
case the correction to be added to the determination of acetone 
from /3-hydroxybutyric acid is 17 instead of 15 per cent (see Table I). 

Table I gives the recovery of the acetone bodies when added 
to blood. These compounds were prepared in the way already 
described in the paper on the determination of the acetone bodies 
in urine, and the percentage of recovery is practically the same 
as that found in the former study. 

Table II gives the values found for determinations carried 
out on the reagents alone. These were carried out in the same 


378 


Acetone Bodies in Blood 


way that has already been described in the paper on urine, except 
for the extra preliminary distillation of both fractions mentioned 
above. There is good agreement among the duplicate determina¬ 
tions even when these are carried out on different days. It is 

TABLE i. 


Recovery of Substances Added to Blood. 


Concentra¬ 
tion pre¬ 
pared. 

Taken. 

Found. 

Calculated 
per 5 cc. 

Added 
per 5 cc. 

Per cent. 

Filtrate. 

| Blood. 





Acetone. 




mg. per 

100 cc. 

cc. 

cc. 

mg. 

mg. 

mg. 


8.6 

50 

2i 

0.233 

0.446 

0.432 

106 

31.0 

50 

2* 

0.735 

1.470 

1.55 

95 

80.1 

50 

2§ 

1.93 

3.8 

4.05 

95 

160.2 

50 

2* 

3.83 

7.66 

8.10 

95 


Acetoacetic acid. 


2.0 

50 

2! 

0.0585 

0.107 

0.101 

106 

5.6 

50 

2§ 

0.143 

0.286 

0.285 

100 

11.3 

50 

2* 

0.255 

0.510 

0.564 

90.5 

61.0 

50 

2\ 

1.54 

3.08 

3.05 

101 

119.4 

50 

2J 

2.96 

5.92 

5.97 

99 


/3-Hydroxybutyric acid. 


0.9 

60 

3 

0.026 

0.0434 

0.0454 

96 

1.8 

60 

3 

0.046 

0.0767 

0.0909 

84 

3.6 

60 

3 

0.089 

0.149 

0.182 

83 

9.1 

60 

3 

0.228 

0.380 

0.4545 

84 

18.2 

60 

3 

0.433 

0.723 

0.909 

80 

18.2 

50 

2\ 

0.385 

0.741 

0.909 

82 

36.4 

50 

2 \ 

0.741 

1.48 

1.82 

82 

44.4 

50 

2* 

0.936 

1.87 

2.27 

82.5 

90.9 

50 

2| 

1.83 

3.65 

4.545 

81 


All calculations are made in terms of acetone. 


not necessary to run a blank on the reagents every time that a 
determination is carried out if the precautions described in the 
preceding paper are observed. In other words, the last flask 
must be freshly boiled out with sodium peroxide, a cork stopper 



































R. S. Hubbard 


379 


must be used in connecting that flask with the still, and, most 
important of all, the laboratory must be free from fumes. 

Tables III and IV show that good agreement on duplicate 
samples of blood can be obtained by this technique. 


TABLE II. 

Blanks on Blood Reagents. 


Date. 

Filtrate. 

Acetone + acetoacetic acid. 

/3-Hydroxybutyric acid. 

Found. • 

Per 50 cc. 
filtrate. 

Found. 

Per 50 cc. 
filtrate. 

19t0 

cc. 

mg. 

mg. 

mg. 

’ mg. 

Nov. 13 

150 

0.0147 

0.0049 

0.0262 

0.0087 

tt 

13 

100 

0.0112 

0.0056 

0.0182 

0.0091 

tt 

13 

50 

0.0082 

0.0082 

0.0107 

0.0107 

tt 

13 

50 

0.0082 

0.0082 

0.0097 

0.0097 

tt 

13 

50 

0.0092 

0.0092 

0.0137 

0.0137 

tt 

13 

50 

0.0067 

0.0067 

0.0072 

0.0072 

tt 

17 

150 

0.0205 

0.0068 

0.0165 

0.0055 

tt 

17 

100 

0.0155 

0.0077 

0.0235 

0.0117 

It 

17 

50 

0.0065 

0.0065 

0.0135 

0.0135 

it 

17 

50 

0.0065 

0.0065 

0.0110 

0.0110 

tt 

17 

50 

0.0125 

0.0125 

0.0095 

0.0095 

tt 

17 

50 

0.0070 

0.0070 

0.0225 

0.0225* 


All results are expressed in terms of the acetone equivalent of the blank. 

When determinations were carried out in the routine manner on 50 cc. 
of filtrate (equivalent to 2.5 cc. of blood) the difference between the most 
widely varying blanks from preformed acetone plus acetoacetic acid is 
equivalent to a difference of 0.1 mg. in 100 cc. of blood; under the same 
conditions the maximum variation for the blank from the /3-hydroxy- 
butyric acid fraction is 0.6 mg. in 100 cc.; if the starred (*) value is omitted, 
this difference of 0.1 and 0.2 mg. is based on eight and on seven separate 
determinations, respectively. 

These blanks were obtained by redistilling the first distillate from 
NaOH, H 2 S0 4 + KMn0 4 , and Na 2 C> 2 , successively. 

It was thought that possibly small amounts of acetoacetic acid 
might be precipitated by the preliminary treatment of blood. 
It was possible that substances in blood might be lost in this way, 
although, when larger amouuts were added, such added amounts 
might be recovered. To test' this possibility a series of bloods 
ranging from an acetone content of 0.05 mg. per 100 cc. to a content 
of 2.5 mg. per 100 cc. was tested by the following technique: 













380 


Acetone Bodies in Blood 


TABLE III. 

-Analysis of p-Hydroxybutyric Acid in Samples of Defibrinated Beef Blood. 


Titration of 
iodine solution. 

Titration 
of distillate. 

Difference. 

Acetone. 

Blank 

equivalent. 

Acetone 
corrected for 
blank. 

cc. 0.01035 n 
thiosulfate 

cc. thiosulfate 

cc. thiosulfate 

mg. 

mg. acetone 

mg. 

9.80 

8.90 

0.90 

0.090 


0.050 

9.80 

8.95 

0.85 

0.085 

0.04 

0.045 

24.60 

23.75 

0.85 

0.085 


0.045 

24.60 

23.65 

0.95 

• 

0.095 


0.055 


TABLE IV. 

iQ-Hydroxybutyric Acid in 100 Cc. Defibrinated Beef Blood. 


Sample taken for analysis. 

Acetone found. 

Corrected for 

80 per cent recovery. 

cc. 

mg. 


5 

3.6 

4.5 

5 

3.6 

4.5 

5 

3.6 

4.5 

5 

3.7 

4.6 

1 

3.6 

4.5 


The sample of blood gave 0.5 mg. of acetone from preformed acetone 
and acetoacetic acid. 


TABLE V. 


Effect of Precipitation on the Determination of Acetone in Blood. 


Blood 

taken. 

Acetone found. 


Taken. 

Acetone found. 




Filtrate. 

Blood. 



cc. 

mg. 

mg. per 
100 cc. 


cc. 

cc. 

mg. 

mg. per 

100 cc. 

5 

0.0605 

1.2 


50 

2* 

0.0240 

1.0 

5 

0.135 

2.7 


50 


0.0695 

2.7 

5 

0.0953 

1.9 


50 

2| 

0.0417 

1.7 

5 

0.0133 

0.3 


50 

2£ 

0.00944 

0.4 

5 

0.142 

2.8 


100 

5 

0.138 

2.8 

5 

0.0020 

0.04 


100 

5 

0.0025 

0.05 

5 

0.0565 

1.1 


100 

5 

0.0497 

1.0 


Results are acetone from preformed acetone plus acetoacetic acid 
expressed in terms of acetone. 

Each pair of determinations was carried out in different bloods. All 
were purified by redistillation as described. 


































R. S. Hubbard 


381 


5 cc. of blood were measured into an 800 cc. Kjeldahl flask, about 
200 cc. of water and 10 cc. of sulfuric acid (1 part of the concen¬ 
trated acid diluted with 1 part of water) added, and the mixture 
was distilled for 20 minutes. The distillate was then purified 
by successive redistillation from sodium hydroxide, sulfuric acid 
plus potassium permanganate, and sodium peroxide. Duplicate 
samples of the same bloods were analyzed by the technique 
described in this paper; that is, they were precipitated and aliquots 
of the filtrate were distilled and redistilled as described. Table V 
contains the results obtained, and shows that there is good agree¬ 
ment between the values from the treated and untreated blood. 
In view of the recovery of acetone and of acetoacetic acid when 
added to blood before precipitation, and of the agreement shown 

TABLE VI. 


Comparison of Blood Acetone by Iodine and by Scott-Wilson Reagent . 


No. 

Taken. 

8sr»nt.+.-Wilsrm rAftflppnf. 

Trirlin** litratinn 

Filtrate. 

Blood. 





1 

2 

cc. 

50 

50 

cc. 

2i 

2* 

mg. 

0.004 

0.008 

mg. per 

100 cc. 

0.2 

0.3 

mg. 

0.005 

0.0094 

mg. per 

100 cc. 

0.2 

0.3 


Acetone from preformed acetone plus acetoacetic acid determined on 
two normal bloods. 


between results on blood and the filtrate from the precipitation, 
it is certain that these compounds are not removed even in small 
amounts by the precipitation with colloidal iron, basic lead acetate, 
and sodium hydroxide. 

The acetone plus acetoacetic acid from two bloods was deter¬ 
mined by both iodine 'titration and precipitation with Scott- 
Wilson’s reagent. The results are given in Table VI, and show 
good agreement between the values obtained. The analysis with 
Scott-Wilson’s reagent was carried out as described in the pre¬ 
ceding paper. 

Table VII presents the results obtained on a series of bloods 
from different types of cases. It contains, in addition to the 
values of the acetone bodies, the values for blood sugar as deter¬ 
mined by the method of Benedict (1918) and of carbon dioxide- 















1 

1 

1 

1 

2 

2 

3 

4 

5 

6 

7 

8 

8 : 

9 

10 

11 

12 

13 

14 

15 

16 

17 

17 

18 

18 

19 

19 

20 

21 

22 

23 

24 

25 

26 

* 

R. 

cpn 

A 1 


Acetone Bodies in Blood 


TABLE VII. 

Blood Determinations . 


Date. 

Acetone 

+ 

aceto¬ 
acetic 
acid 
express¬ 
ed as 
acetone. 

^-Hy¬ 

droxy- 

butyric 

acid. 

Sugar. 

Alkali 

re¬ 

serve. 

Remarks. 


mg. per 
100 cc. 

mg. per 
100 cc. 

per cent 

vol. per 
cent 


Nov. 19, 1919 

0.3 

0.8 



Normal. 

Dec. 8, 1919 

0.1 

0.1 

0.098 

65.3 


Jan. ?, 1920 

0.1 

0.4 




Nov. 11, 1920 


0.4 

0.098 

74.0 


Dec. 17, 1919 

0.1 

0.3 

0.121 

71.0 

Normal. 

Nov. 18, 1920 

0.7 

0.4 

0.135 

68.3 


Dec. 14, 1919 

0.3 

0.4 

0.125 

76.8 

Normal. 

Nov. 27, 1920 

0.3 

0.4 

0.102 

78.0 

CC 

Dec. 3, 1920 

0.2 

0.3 

0.125 

61.7 

CC 

Nov. 5, 1919 

0.8 


0.118 

61.5 

CC 

Dec. 10, 1919 

0.6 

0.7 

0.111 

56.8 

Obese. 

Feb. .17, 1921 

0.9 

0.02 

0.139 

58.7 

Arthritic. 

“ 20, 1921 

1.8 

2.9 

0.153 

51.0 


Nov.' 24, 1920 

0.6 

0.4 

0.128 

50.4 

Nephritic. 

Dec. 18, 1920 

1.4 

1.6 

0.167 

46.2 

CC 

Nov. 6, 1920 

1.1 

0.8 


62.4 

Gastroin¬ 






testinal. 

Dec. 11, 1919 

0.7 

2.5 

0.098 

56.0 

Thyroid. 

“ 10, 1919 

0.03 

0.2 

0.102 


(( 

Nov. 29, 1919 

0.1 

0.6 

O'. 109 

66.0 

u 

Dec. 7, 1919 

0.2 

0.3 

0.125 

78.7 

« 

Nov. 24, 1920 

0.8 

0.9 

0.222 

72.1 

Diabetic. 

Dec. 17, 1919 

0.8 

0.6 

0.190 

50.0 

CC 

Jan. 9, 1920 

1.2 

1.4 

0.125 

67.2 


Dec. 4, 1920 

6.5 

8.9 

0.222 

42.8 

Diabetic. 

“ 18, 1920 

2.7 

5.2 

0.154 



“ 10, 1920 

1.0 

1.0. 

0.250 

57.9 

Diabetic. 

“ 18, 1920 

1.2 

3.1 

0.190 



“ 7, 1920 

1.0 

2.4 

0.161 

57.6 

Diabetic. 

Nov. 11, 1919 

0.0 

0.4 

0.266 


u 

“ 11, 1919 

0.9 

0.0 

0.144 


u 

Jan. 10, 1920 

2.8 

3.7 

0.185 


(( 

Aug. 18, 1919 

0.7 

2.0 

0.128 

57.0 

u 

“ 18, 1919 

1.5 

1.5 

0.092 


u 

Dec. 5, 1920 

0.8 

0.7 

0.156 

48.5 

(( 


rkedly high in fat fed for 4 days. 

stone, acetoacetic acid, and /3-hydroxybutyric acid are 


3 measured as the C02-combining capacity of the plasma. 
















R. S. Hubbard 


383 


combining power of the plasma as determined by the method of 
Van Slyke and Cullen (Van Slyke, 1917, a; Van Slyke and Cullen, 
1917). The bloods analyzed were kept from clotting with potas¬ 
sium oxalate, and were analyzed on the same day that they were 
taken. In most cases the bloods were drawn before breakfast. 
Inspection of the table shows that the values for normal bloods 
range from 0.1 to about 1.0 for acetone from all three acetone 
bodies. Results on cases of diabetes sometimes fall into the same 
range, and are sometimes much higher. Neither in normal nor 
in pathological specimens is there any relationship between the 
amount of acetone from acetone plus acetoacetic acid and that 
from jS-hydroxybutyric acid except in the case of diabetic bloods 
in which both values are high; in them the acetone from 0- 
hydroxybutyric acid is in excess. 

CONCLUSION. 

A method for the determination of the acetone bodies in blood 
has been described which gives a high and constant percentage of 
recovery for added acetone bodies, and which gives good agree¬ 
ment between duplicate determinations. Agreement is also 
found between the values of the acetone as determined by two 
different methods—a fact which renders it probable that the 
substance so determined is acetone. The results obtained by 
this method on blood from normal subjects are low. The 
accuracy of the determination is about 0.1 mg. per 100 cc. of 
blood. 

My thanks are due to various members of the Staff of The 
Clifton Springs Sanitarium for the pathological specimens of 
blood analyzed. 

BIBLIOGRAPHY. 

Benedict, S. R., J. Biol. Chem., 1918, xxxiv, 203. 

Hubbard, R. S., /. Biol. Chem., 1920, xliii, 43. 

Hubbard, R. S., J. Biol. Chem., 1921, xlix, 357. 

Hubbard, R. S., and Wright, F. R., J. Biol. Chem., 1921, xlvi, p.xiii. 
Kennaway, E. L., Biochem. J., 1914, viii, 230. 

Kennaway, E. L., Biochem. J., 1918, xii, 120. 

Ljungdahl, M., Biochem. Z., 1917, lxxxiii, 103. 


384 


Acetone Bodies in Blood 


Ljungdahl, M., Biochem. Z., 1919, xciii, 325. 

Marriott, W. McK., J. Biol. Chem., 1913-14, a, xvi, 281. 

Marriott, W. McK., J. Biol. Chem., 1913-14, b, xvi, 289. 

Marriott, W. McK., J. Biol. Chem., 1914, a, xviii, 241. 

Marriott, W. McK., J. Biol. Chem., 1914, b, xviii, 507. 

Scott-Wilson, H., J. Physiol., 1911, xlii, 444. 

Short, J. J., J. Biol. Chem., 1920, xli, 503. 

Van Slyke, D. D., J. Biol. Chem., 1917, a, xxx, 347. 

Van Slyke, D. D., J. Biol. Chem., 1917, b, xxxii, 455. 

Van Slyke, D. D., and Cullen, G. E., J. Biol. Chem., 1917, xxx, 289. 
Van Slyke, D. D., and Fitz, R., J. Biol. Chem., 1917, xxxii, 495. 
Van Slyke, D. D., and Fitz, R., J. Biol. Chem., 1920, xxxix, 23. 
Widmark, E. M. P., Biochem. J., 1919, xiii, 430. 


Reprinted from The Journal of Biological Chemistry 
Vol. xlix, No. 2, December, 1921 


BLOOD ACETONE BODIES AFTER THE INJECTION OF 
SMALL AMOUNTS OF ADRENALIN CHLORIDE.* 

By ROGER S. HUBBARD and FLOYD R. WRIGHT. 

C From the Clifton Springs Sanitarium , Clijton Springs, New York.) 

(Received for publication, October 4,1921.) 

In an earlier paper (Hubbard, 1921) results were reported on 
the study of normal subjects under conditions which caused a 
slightly increased excretion of the ^cetone bodies. The results 
showed that under such conditions there was an amount of aceto- 
acetic acid excreted during the development of acetonuria which 
was in excess of the /3-hydroxybutyric acid simultaneously ex¬ 
creted. Conclusions from these findings were uncertain, as the 
results were complicated by differences in the kidney thresholds 
of the different compounds, and these prevented certain inter¬ 
pretation of conditions within the organism. For this reason 
it seemed desirable to investigate conditions other than dietary 
changes which might give rise to increased production of the ace¬ 
tone bodies, and in which the increase and return to normal 
values would take place within a comparatively short period of 
time. The effect of the injection of adrenalin chloride was 
selected for study. 

Peters and Geyelin (1917) have reported experiments which 
showed that after the injection of adrenalin chloride solution 
there were changes in the carbon dioxide-combining capacity of 
the plasma as well as in the blood sugar content and blood pressure. 
These experiments indicate that there are extensive changes in 
the chemistry of the blood brought about by the presence of large 
amounts of this substance, and it was thought that changes might 
be found in the acetone bodies. Eiselt (1910) has reported an 
increase in urine acetone as the result of the injection of adrenalin 
into a patient suffering from Addison’s disease. 

*A preliminary report of the work described below was given before 
the American Society of Biological Chemists, in December, 1920 (Hubbard 
and Wright, 1921). 


385 


386 


Blood Acetone Bodies 


A series of seven experiments was run on normal men. Each 
subject was fed a standard simple breakfast, and an hour after¬ 
wards received an injection of 0.5 or 1 cc. solution of adrenalin 
chloride in a one to one thousand dilution. A sample of blood 
was taken before the injection was given, and other samples were 
taken at various intervals after the injection. Each sample was 
analyzed for acetone from preformed acetone plus . acetoacetic 
acid and for acetone from /3-hydroxybutyric acid by the tech¬ 
nique described in the preceding paper (Hubbard, 1921), for sugar 
by the technique described by Benedict (1918), and for the car¬ 
bon dioxide-combining capacity of the plasma by the method 
of Van Slyke and Cullen (Van Slyke, 1917; and Van Slyke and 
Cullen, 1917). Changes in the systolic and diastolic blood pres¬ 
sure and the pulse rate were also recorded. These last showed no 
anomalies, except the expected variations with the larger dose of 
adrenalin chloride, and a detailed description of them has accord¬ 
ingly been omitted from this paper. 

The results obtained from the chemical analysis of the various 
samples of blood on the different patients are listed in Table I. 
Three of the experiments show a distinct rise of the acetone bodies, 
with a subsequent return to the values found preceding the 
injection of the adrenalin. In two of these cases both of the frac¬ 
tions show the increase. The experiments which showed the 
most marked variations are experiments in which the subject 
received a dose of 1 cc. of the drug. There was one experiment 
in which a dose of 1 cc. was given, and in which there was no 
change in the acetone bodies. The subject of this experiment 
showed a high blood sugar value in the sample taken before the 
injection and perhaps should not be classed as normal but there is 
no other reason for making such’an assumption. There is no 
constant relationship in the degree of response of the different 
acetone bodies, and the results in the cases where there is a rise 
noted do not seem to bear any relationship to the changes in 
blood sugar nor in the carbon dioxide-combining power of the 
plasma. The magnitude of the rise observed in some cases and 
the subsequent return to normal values indicate that the changes 
are real changes in the substances present in the blood, induced 
by the adrenalin chloride administered. 


R. S. Hubbard and F. R. Wright 


387 


.TABLE I. 


Case 

No. 

Sex. 

Date. 

Dose 

Time. 

Acetone 
plus 
aceto- 
acetic 
acid per 
100 cc. 

0-Hy- 
droxy- 
butyric 
acid per 
100 cc. 

Sugar. 

Plasma 

bicar¬ 

bonate 

COs. 




cc. 

hrs. 

mg. 

mg. 

per cent 

vol. 

''per cent 

1 

Male. 

Dec. 8, 1919 

i 

Before. 

0.1 

0.1 

0.098 

65.3 






0.3 

0.3 

0.156 






n 

0.05 

0.2 

0.160 

72.4 





2a 



0.100 

71.0 

1 

u 

Nov. 7, 1920 

l 

Before. 


0.25 

0.098 

74.0 





\ 

0.6 

1.1 

0.167 






1 

1.3 

1.7 

0.225 

53.8 





2 

0.5 

0.2 

0.136 

51.0 

2 

u 

Dec. 17, 1919 


Before. 

0.1 

0.3 

0.121 

71.0 





\ 

0.1 

0.3 

0.133 

71.0 





11 

0.25 

0.4 

0.222 

69.1 





2^ 

0.1 

0.4 

0.160 

71.0 

2 

« 

Nov. 18, 1920 

l 

Before. 



0.135 

68.3 





\ 


0.3 

0.157 

59.8 





1 

0.2 

0.0 

0.215 

56.6 



- 


2 

0.2 

0.2 

0.194 

55.1 

3 

u 

“ 14, 1919 

h 

Before. 

0.3 

0.4 

0.125 

76.8 





\ 

0.5 

1.1 

0.154 

76.8 





11 

0.6 

0.5 

0.128 

67.3 





2i 

0.25 

0.35 

0.122 

76.8 

5 

it 

Dec. 3, 1920 

1 

Before. 

0.2 

0.3 

0.125 

61.7 





* 

0.3 

0.3 

0.154 

62.6 





1 

0.6 

0.1 

0.200 

62.1 





2 

0.3 

0.3 

0.122 

59.8 

4 

u 

Nov. 27, 1920 

1 

Before. 

0.3 

0.4 

0.102 

65.3 



• 



0.8 

0.5 

0.236 

59.5 





1 

0.8 

0.9 

0.266 

55.7 





2 

0.3 

0.3 

0.166 

61.4 


Results for all three acetone bodies are expressed as acetone. 

Alkaline reserve is measured as the C0 2 -combining capacity of the 
plasma. 
















388 


Blood Acetone Bodies 


The most satisfactory explanation for the results reported is 
in the probable local restrictions of the blood supply induced by 
the drug, which lead to a local production—or failure of combus¬ 
tion—of the acetone bodies. Such production could occur under 
these conditions in spite of the increased glucose content of the 
blood. 

The production of these acetone bodies certainly cannot be 
looked upon as responsible in any degree for the marked lowering 
of the alkaline reserve observed. 

The experiments do not afford any information concerning 
the question of the order of the production of the acetone bodies 
in the organism. 

Our thanks are due to the members of the Staff of The Clifton 
Springs Sanitarium who served as subjects for these experiments, 
and particularly to the late Dr. Malcolm S. Woodbury, superin¬ 
tendent of the Sanitarium, for the continued encouragement which 
he extended to us during our experiments. 

BIBLIOGRAPHY. 

Benedict, S. R., J. Biol. Chern., 1918, xxxiv, 203. 

Eiselt, R., Z. klin. Med., 1910, lxix, 393. 

Hubbard, R. S., J. Biol. Chem., 1921, xlix, 375. 

Hubbard, R. S., and Wright, F. R., J. Biol. Chem., 1921, xlvi, p. xiii. 
Peters, J. P., Jr., and Geyelin, H. R., J. Biol. Chem., 1917, xxxi, 471. 

Van Slyke, D. D., J. Biol. Chem., 1917, xxx, 347. 

Van Slyke, D. D., and Cullen, G. E., J. Biol. Chem., 1917, xxx, 289. 


Reprinted from The Journal of Biological Chemistry 
Vol. L, No. 2, February, 1922 


STUDIES ON THE ACETONURIA PRODUCED BY DIETS 
CONTAINING LARGE AMOUNTS OF FAT.* 

By ROGER S. HUBBARD and FLOYD R. WRIGHT. 

{From the Laboratories of The Clifton Springs Sanitarium, Clifton Springs, 
New York.) 

(Received for publication, December 3, 1921.) 

The excretion of the acetone bodies—acetone, acetoacetic 
acid, and /3-hy droxy butyric acid—in conditions in which the 
organism is not utilizing carbohydrate either through a de¬ 
ficiency of foodstuff of this kind in the diet or through the inability 
of the organism to metabolize the food when supplied, as in dia¬ 
betes mellitus, has attracted attention for many years, and a 
large amount of literature has collected on the subject. In two 
papers recently published, Shaffer (1921, a, b) has summarized 
this literature, and has suggested certain methods of studying the 
problem which are somewhat different from those which have 
been used before. In this paper are reported some experiments 
which were carried out along the lines suggested, and which appear 
to support the theses advanced in these two articles. 

In his first paper Shaffer (1921, a) reported experiments on the 
oxidation of mixtures of acetoacetic acid and glucose by alkaline 
hydrogen peroxide which showed that if there were present in 
the mixture one molecule or more of glucose for each molecule of 
acetoacetic acid, the acid was oxidized under suitable conditions 
of temperature, alkalinity, etc., but that if the relative concen¬ 
tration of glucose was less than this, the oxidation of the keto- 
acid was not as complete. In the second paper (Shaffer, 1921, b) 

* A preliminary report of the clinical side of the work discussed was read 
before the meeting of the New York State Medical Association in Brooklyn, 
May, 1921, by Floyd R. Wright; a portion of the work formed part of a thesis 
presented for partial fulfilment of the requirement for the degree of Doctor 
of Philosophy at Washington University, St. Louis, in June, 1921, by Roger 
S. Hubbard. 


361 


362 


Studies on Acetonuria 


he studied the problem from the point of view of the metabolism 
of human subjects, and concluded that a reaction of a similar 
nature takes place in the body. Since the appearance of these 
two articles Woodyatt (1921) has published a paper in which the 
subject is discussed from the standpoint of the practical treat¬ 
ment of diabetes, and in which data are presented which support 
the conclusions stated above. 

The theory which has been developed in these papers, and on 
which the following paper is based, is that acetoacetic acid itself 
is not easily burned in the body, but that it forms with glucose, 
or with degradation products of glucose and related substances, 
a compound which is easily burned. To compounds which give 
rise, in the progress of metabolism, to acetoacetic acid the name 
“ketogenic” is given, while the name “ antiketogenic” has been 
applied to compounds which furnish glucose or other related 
compounds with which the acetone body combines. The keto¬ 
genic compounds contained in the diets are the fatty acids con¬ 
tained in the fats and the a-amino-acids, leucine, tyrosine, phenyl¬ 
alanine, and possibly histidine which forms a part of the proteins. 
There is probably a molecule of the acetone bodies derived from 
each molecule of these compounds contained in the diet. 

The amounts and source of the antiketogenic compounds con¬ 
tained in the diet are more uncertain. Glucose and related sugars, 
as levulose, form one source of these substances, whether taken 
as the sugars themselves or as the more complex carbohydrates. 
Protein yields glucose when fed to the total diabetic in amounts 
which vary with the different kinds of the foodstuff, and some 
percentage of the protein should therefore be included with the 
carbohydrate in figuring the total antiketogenic intake. There 
is, too, considerable data which indicate that glycerol yields glu¬ 
cose under some conditions, and so fat, from which glycerol is 
produced by hydrolysis in the organism must also be considered 
as a possible source of antiketogenic compounds. 

The question is even more complicated than this, because it is 
not certain what derivatives of the glucose-forming a-amino-acids 
and glycerol act as antiketogenic compounds. Shaffer (1921, b) 
has pointed out this difficulty clearly. He states: 1 


1 Shaffer (1921, b), p. 458. 


R. S. Hubbard and F. R. Wright 


363 


“ • • • • the two carbon residues from glycocoll and the three carbon 
residues from the other sugar-forming amino-acids may have direct and 
immediate antiketogenic (ketolytic) action without condensation to glu¬ 
cose, and the same may be true of glycerol.” 

To determine the border-line diet which should just produce an 
excretion of the acetone bodies Shaffer (1921, 6) calculated the 
molecular equivalents of the ketogenic compounds from fat and 
protein, and of the antiketogenic compounds from carbohydrate, 
protein, and the glycerol residue of the fat to the extent of the 
glucose which could be derived from them. From the analysis 
of the data so obtained he concluded that a diet containing 10 
per cent of the calories in the form of protein, 10 per cent as car¬ 
bohydrate, and 80 per cent as fat represented approximately the 
border-line diet. He studied this diet in the light of data con¬ 
tained in the literature and obtained experimentally, and showed 
that the theory was confirmed by such results as were available. 

In the experiments reported in this paper an attempt was made 
to study this diet described by Shaffer, and diets in which the 
relative amounts of carbohydrate and fat were somewhat varied. 
To obtain a method of graphic representation of the various diets 
in terms of their “ketogenic balance” the following plan was 
adopted. The excess antiketogenic material derivable from pro¬ 
tein, that is, the amount of glucose which protein would yield 
greater than that needed to bring about oxidation of the keto¬ 
genic material from the same protein, was calculated from the 
data presented by Shaffer (1921, b). He showed from the analyses 
of the a-amino-acid content of ox muscle given by Lusk (1917) 2 
and from the glucogenetic power of protein, that there are twice 
as many gram molecules of antiketogenic substance (glucose) 
as of ketogenic compounds which can be derived from a given 
weight of this protein. Glucose is derived from ox muscle at the 
rate of 58 gm. for each 100 gm. of the protein ingested (Woodyatt, 
1921), and there will be 29 gm. of excess glucose for each 100 gm. 
of this protein fed. The amounts of glucose and of acetoacetic 
acid which can be derived from different proteins vary, and 25 
gm. have been chosen as a convenient average figure to express 
the amount of glucose available for additional antiketogenic 


2 Lusk (1917), p. 77. 


364 


Studies on Aceionuria 


action from 100 gm. of protein. This calculation is similar to 
that suggested by Woodyatt (1921). To the excess glucose from 
protein was added the glucose taken in the diet, and the sum was 
multiplied by 1.5 (molecular weight of glucose = 180; molecular 
weight of stearic acid = 284; of palmitic acid = 256; of oleic acid = 
270 

282; average = 270; — = 1.5) to convert the result into terms 

lot) 

of its fatty acid molecular equivalent. This product was divided 
by the fatty acid content of the diet (95 per cent of the fat) and 
the ratio was multiplied by 100 to give the resulting expression 
in the form of per cent. 

This formula for expressing the “ketogenic balance” of any diet 
is expressed as follows: 

1.5 (weight glucose + 25 per cent weight protein) 

95 per cent weight fat 

In preparing the charts in this paper the total carbohydrate 
content of the diet has been used instead of the glucose content. 
Such a substitution introduces an error, as the intake of starch 
in grams should be multiplied by 1.1 to give the correct amount 
of glucose to which it is equivalent, but the difference between 
the values is almost certainly within the limit of error, and the 
total carbohydrate content is more easily calculated from published 
tables. 

Before proceeding to a study of the experiments, attention must 
be called to some of the limitations and advantages of the formula 
given above. In the first place it is based on an assumption 
which did not hold exactly for any of the diets studied. The 
formula assumes that all of the fat contained in the diets was fed 
in the form of glycerides of the higher fatty acids—palmitic, 
stearic, and oleic—and such a diet could not be fed for any con¬ 
siderable period. In one of the experiments an attempt was made 
to approximate such a composition for a few days, as will be de¬ 
scribed below, but for the most part butter and cream formed a 
large percentage of the fat ingested. These fats contain rela¬ 
tively large amounts (up to about 8 or 9 per cent) of their fatty 
acids in the form of compounds of comparatively low molecular 
weight, and, therefore, yield more ketogenic material per gram 
than do fats not derived from milk. In the formula a figure 



R. S. Hubbard and F. R. Wright 


365 


lower than 1.5 should be used to convert antiketogenic compounds 
expressed as glucose into molecular equivalents of fatty acids 
when these fats are included in the diet. Butyric acid, forex- 
ample, has a molecular weight of 88, and if tributyrin were the 
only fat fed the sum of the antiketogenic compounds expressed 
as glucose should be multiplied by 0.49 ( T 8 A) to express the 
fraction in terms of relative molecular concentrations. However, 
this error will not change the numerical value of the expression 
by more than 5 per cent; the error introduced by figuring from 
the carbohydrate content of the diet was of the same order of 
magnitude, and the two should practically compensate for each 
other. 

The second objection to the formula has been indicated already. 
It is impossible to be sure that the figure used to express the ex¬ 
cess antiketogenic material from protein is correct. The value 
will vary for different proteins as their content of leucine, tyro¬ 
sine, and phenylalanine varies, and will also vary because the 
glucogenic power of different proteins is different. The effect of 
this uncertainty upon the numerical expression is illustrated by 
the following figures. In a diet in which 10 per cent of the calories 
is fed as protein, 10 per cent as carbohydrate, and 80 per cent as 
fat the numerical value of the expression given above is 55 per 
cent. The values of similar expressions in which different figures 
express the excess glucose from protein would vary from 31 per 
cent if its antiketogenic power is neglected, to 71 per cent if its 
ketogenic power is neglected, and the glucose which can be de¬ 
rived from protein is figured at 60 per cent of the total weight. 
If protein contains both ketogenic and antiketogenic materials— 
and this is almost certainly the case—the different figures lie well 
within the limits of error with which such formulas can be applied 
to the study of actual diets. 

In case the antiketogenic effect of protein depends, not on 
glucose, but on the two and three carbon atom residues derived 
from the sugar-forming a-amino-acids the values of the expres¬ 
sion would be much higher than 71 per cent. In that case an 
a-amino-acid would figure three times as efficient as glucose if 
a two carbon atom residue takes part in the reaction as an anti¬ 
ketogenic compound, or twice as efficient if a three carbon atom 
residue takes such a part. It seems almost certain that it would 


THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. L, NO. 2 


366 


Studies on Acetonuria 


be possible to detect such a marked effect as this experimentally, 
and an attempt has been made to interpret the data presented 
below in such a way as to solve this problem. 

In the formula given no account has been taken of the 
possible antiketogenic effect of the glycerol radicle present in 
the fats. This radicle is probably the most uncertain of the 
different possible sources of antiketogenic compounds contained 
in the diet, and the part which it plays in the reaction can 
be better discussed after a study of the data obtained experi¬ 
mentally. If different diets are fed, and the degree of acetonuria 
is noted and compared with some such numerical expression 
of their ketogenic balance as that suggested, the value of the ratio 
at which the excretion of the acetone bodies becomes normal will 
represent the condition of ketogenic antiketogenic equilibrium. 
From the numerical value of this ratio it should be possible to 
determine whether the glycerol residue figures as a source of anti¬ 
ketogenic compounds. If this residue yields glucose in the organ¬ 
ism, and this glucose acts as an antiketogenic compound, enough 
of such material would be furnished in each gram of fat to combine 
with one-sixth of the ketogenic material, so that only five-sixths 
of the total fatty acids will be free to combine with the antiketo¬ 
genic compounds from carbohydrate and protein; in this case 
acetonuria should develop and clear up when the diet has a value 
of 83 per cent. If glycerol in fat does not produce antiketogenic 
compounds in the organism, all of the fatty acid will take part in 
the reaction with antiketogenic material from other foods, and the 
border-line diet will have a numerical value of 100 per cent. If 
glycerol figures as an antiketogenic compound in the form of a 
three carbon atom residue, one-third of the total fatty acid will 
combine with it, two-thirds of it must be burned by the help of 
other foodstuffs, and the border-line diet should have a value of 
67 per cent. In the experiments reported here an attempt has 
been made to make such comparisons, and to determine whether 
the glycerol residue of fat does possess an antiketogenic action. 

The study of diets which produce border-line acetonuria and 
at the same time maintain the body weight of the subjects is 
rather difficult. The diets are markedly different from those 
generally eaten, and many patients return a portion of the fat 
untouched. There is also a temptation to break the dietary 


367 


R. S. Hubbard and F. R. Wright 

restrictions not unlike the temptation to which diabetics are sub¬ 
ject, and it would not be necessary for a patient to ingest much 
more carbohydrate than is furnished to spoil an experiment. In 
the series presented here only such cases are included as could be 
studied under rather close supervision in a department devoted 
exclusively to the study of nutritional diseases. We wish here 
to express our thanks to Dr. S. T. Nicholson, Jr., the director 
of this department, and the dietitians and nurses attached to it 
for their cooperation in our experimental work. 

The series included two experiments on a normal subject, one 
of which has been previously reported in another connection, and 
studies on four cases of arthritis who were undergoing the dietary 
treatment recommended by Pemberton (1917) in which the car¬ 
bohydrate intake is reduced. These cases can probably be con¬ 
sidered as normals for the purpose of such a study, although 
Pemberton and Foster (1920) have stated that such patients 
show a slightly increased concentration of sugar in the blood and 
an abnormal rise in blood sugar after the administration of large 
doses of glucose. 

In all of the experiments the attempt was made to furnish 
enough food to each patient to maintain the body weight un¬ 
changed. To accomplish this the basal metabolism was deter¬ 
mined with the Benedict portable respiration calorimeter (Bene¬ 
dict, 1918) and enough calories were fed to allow for the main¬ 
tenance of basal equilibrium and for the probable activity of the 
patient. Usually the food provided for a bed patient was so 
calculated as to furnish 20 to 25 per cent more calories than his 
basal requirement called for, and this was found to be satisfac¬ 
tory for most of the subjects. It is desirable that the patients 
should be in nitrogen as well as in metabolic equilibrium, and at 
the same time that the protein content of the diet should be kept 
low to diminish the uncertain factor of its part in the ketogenic 
expression. In the experiments reported 10 per cent of the 
total calories were fed as protein in most of the diets studied. 
The relationship between the nitrogen intake and the output of 
nitrogen in the urine was determined, and it was found that there 
was little difference between them. If the excess antiketogenic 
compound had been figured from the urinary nitrogen instead of 
from the protein intake, the value of the ratio described would 


368 


Studies on Acetonuria 


not have varied beyond the limits of experimental error. The 
intake of carbohydrate and of fat formed, respectively, the sources 
of 10 and of 80 per cent of the calories in the basal diet, and of 
varying percentages—5 and 85 per cent, 15 and 75, 20 and 70 per 
cent—in the other diets studied. An attempt was made to feed 
each of these diets for a period long enough to determine the 
level of acetone excretion which corresponded to it, but it was 
usually necessary to change the more severe diets before such an 
equilibrium was established. 

The diets used were figured from the tables given in Joslin’s 
Diabetic Manual (Joslin, 1919); they were prepared under the 
direction of a competent dietitian, and food not eaten was weighed, 
and the proper allowance made in the record; a complete sample 
diet is given for one of the cases. While a majority of the patients 
ate the diets as furnished, two did not, and the results of the 
studies carried out on them are accordingly not wholly satisfac¬ 
tory. It has seemed best to include these cases in this report, 
however, as they serve as a check upon the results obtained upon 
other subjects. 

The urines were sent to the laboratory daily. It was impossi¬ 
ble to control the completeness of the collection through creatinine 
determinations because the presence of the acetone bodies in 
urine interferes with the method of analysis (Morris, 1915), and 
all of the cases except one showed a large excretion of acetone on 
all of the more severe diets fed. This lack of suitable control of 
the accuracy of collection made it seem best to record and plot 
the concentration of the acetone bodies as well as their total 
excretion. In some specimens marked variations in volume, 
total nitrogen content, and ammonia nitrogen content almost 
certainly show failure to collect accurate 24 hour specimens. 

These daily urines were analyzed for acetone bodies by a method 
recently described (Hubbard, 1921) by which the acetone plus 
acetoacetic acid were determined together as acetone, and the 
/3-hy droxy butyric acid was determined separately, also as acetone. 
Total nitrogen was determined by the direct Nesslerization 
method of Folin and Denis (1916), slightly modified to permit 
the use of the oxidizing and Nessler’s reagents, described by Folin 
and Wu (1919). Ammonia determinations were made by the 
permutit method of Folin and Bell (1917). In some instances 


R. S. Hubbard and F. R. Wright 


369 


other factors were studied which were connected more indirectly 
with the main problem. Total acidity was determined in the 
urine of two of the patients by the method described in Folin’s 
Manual (Folin, 1916), 3 and its hydrogen ion concentration in 
one of the experiments by a colorimetric method using the stand¬ 
ard universal buffer solution described by Acree, Mellon, Avery,, 
and Slagel (1921). This solution was standardized before use 
against phosphate solutions of known hydrogen ion concentra¬ 
tion. Besides these determinations on the urine the stripped 
weight of the patients was recorded, and in most instances the 
tension of carbon dioxide in the alveolar air was estimated. This 
determination was carried out in Cases II and III by the method 
of Marriott (1916) and in the other cases by the Fridericia method 
(Fridericia, 1914; Poulton, 1915). 

The results obtained are given in Tables I to VI, and plotted 
in Charts 1 to 6. In these charts the diet is indicated at the top 
in terms of both total food and of percentage of the calories fur¬ 
nished by the three main classes of foodstuffs. When the diet 
varied much from day to day the average intake was made the 
basis of this plot. The numerical value of the expression 

1.5 (weight carbohydrate) + 25 per cent (weight protein) _ er cen {. 

95 per cent weight fat ^ er ° en 

was plotted to correspond with the intake of food for each day, 
and the daily excretion of all the acetone bodies reckoned as the 
sum of the acetone which could be formed from them was plotted 
below it. Since slight increases of acetone above normal may 
be of considerable importance in this study, the plan was adopted, 
in two of the cases studied, of plotting the excretion of acetone 
bodies in terms of their concentration in the urine upon paper 
with logarithmic characteristics. This method shows differences 
which are actually slight but which may figure as large percentage 
increases because the normal amounts are small. In these plots 
the two fractions of the acetone bodies—acetone plus acetoacetic 
acid and /3-hydroxybutyric acid—were plotted separately to show 
the relationship between the two fractions, and both were plotted 
as acetone to make the curves comparable. The concentrations 


3 Folin (1916), p. 103. 



370 


Studies on Acetonuria 


instead of the total excretions were used because of occasional 
failure in the collection of accurate 24 hour specimens. 

Cases I and IV are reports of two experiments carried out on the same 
normal subject (one of the authors, K.S.H.). The data reported under 
the heading ‘‘Case I” have been previously presented (Hubbard, 1921) 
and are repeated here because a comparison with those obtained on the same 
subject in a later experiment show some things which are not as well 
brought out in other studies. The subject was a man 5 ft. 104 in. tall, who 
weighed 165 lbs. and who at the time of the first experiment was 28 years 
old. During both of the periods he did light laboratory work while the 
experiments were going on. The first series of results was obtained before 
the appearance of Shaffer’s papers in 1921 and the diet was differently 
planned from those used in the other experiments. The fat and carbohy¬ 
drate were fed in different relative amounts—multiples of 50 gm., as a study 
of the table shows—and an attempt was made to feed sufficient protein to 
keep the caloric intake constant. The subject had been living on a normal 
mixed diet up to the first day of the experiment. There was a slight nega¬ 
tive nitrogen balance during the first part of it, and a slight positive one 
after the diet had become more nearly normal, but the difference was not 
great enough in either case to affect the calculation of the probable keto- 
genic balance seriously. The development and clearing up of acetonuria 
is clearly shown in Table I and Chart 1. There was certainly an increased 
acetonuria on a diet which contained 250 gm. of fat, 50 gm. of carbohydrate, 
and 68 gm. of protein; this diet has a ketogenic balance of 42 per cent in 
terms of the formula suggested for expressing that balance. The excretion 
of the acetone bodies was slightly increased during the first 3 days of the 
experiment, but the increases were so slight that they cannot be attributed 
with certainty to the diet. The acetonuria completely cleared up when a 
diet having a ketogenic balance of 152 per cent was fed, and it seems certain 
that the border-line diet, that is, the diet representing ketogenic equilib¬ 
rium, must lie between the two extreme diets fed, and probably does not 
lie far from that fed at the start of the experiment which has a ketogenic 
balance of 97 per cent. Attention should be called to the gradual increase 
and decrease of acetonuria as it developed and cleared up; a study of the 
table and chart makes it seem improbable that the second diet was fed 
long enough to cause a maximum excretion of acetone to correspond with 
its composition. 

The experiment recorded under the heading “Case IV” was carried out 
on the same subject as was that recorded under “Case I.” The height and 
weight were approximately the same as those given in the preceding para¬ 
graph, and the age was 33 years. The basal metabolism measured 1,750 
calories per day. The diets were calculated and fed as here described, 
and the subject ate the entire amount of every diet provided. The collec¬ 
tion of urine samples was accurate. There was some loss of weight during 
the first part of the experiment, but when diets were fed which caused an 


R. S. Hubbard and F. R. Wright 


371 


W 

>-3 


eo 

e 

O 


m 

s 


o 

(H 

fa 


in 

-t-J 

U3 

Q 


N CO ^ «5 O h 


OJ Ui 

OSs.N.V.S.'. 
>eH ^ ^ ^ ^ ^ 


o 


o 

04 

04 

CO 

04 


04 

rH 

05 

cO 

tF 

"C 

>> 

■H 

S 

C3> 

04 

O 

O 

CO 

o 

o 

04 

CO 

no 

04 

TF 

no 

CO 

§ 

04 

O 

04 

O 

04 

o 

3 


d 

O 

o 

d 

O 

rH 

d 

o 

d 

O 

d 














h3s 














d 












o 

•9 

>> 

05 

o> 

>-« 

2.2 

4.4 

no 

CO 

00 

GO 

O 

C5 

04 

05 

no 

5.8 

tF 

co 

tF 

X 

o 

tF 

*c! 

\ 




rH 

CO 

rH 

CO 






d> 

s 






rH 






d 


CO 

oo 

M2 

tF 

CO 

04 

TF 


CO 

X 

tF 

h 

s' 

o 

TF 

CO 


l- 

o 

i> 

o 

04 

04 

04 

a> 

o 

o 

o 

co 

TF 

CO 

04 

rH 

O 

O 

o 


£x> 










"3 


d 

o 

d 

o 

d 

d 

d 

o 

d 

d 

d 

+ 

d 












<D 













G 

o> 

o> 

1> 

tF 

04 

04 

o 

o 

o 

X 

X 

X 

tF 

h> 

<D 

>*H 

\ 

d 

TF 

1> 

tF 

x 

OO 

X 

d 

CO 

04 

rH 

O 

d> 




CO 

no 

cO 

04 

rH 



<5 

s 











£ 


GO 

co 

CO 

b- 

no 

T*H 

tF 

05 

o 

r- 

o 

TJ« 

s' 

ca> 

tF 

tF 


GO 

rH 

1—1 

CO 



CO 

05 

w 

5s 

t> 

o 

4> 

d 

d 

b- 

o 

GO 

o 

05 

d 

05 

d 

X 

d 

co 

d 

CO 

d 

TF 

o 

( 










no 


tF 

<S . 

ojz; 

s' 

no 

o 

CO 

oo 

co 

no 

TF 

no 


co 

05 

H 


04 

r- 

CO 

b~ 


04 

CO 

rH 

05 

o 

05 



rH 

rH 

rH 

rH 

rH 

rH 

rH 

rH 


rH 


d 

s 


no 

0 

no 

no 

no 

o 

o 

o 

o 

o 

o 

• 

CO 

o 

O 

03 

rH 

00 

X 

I 

co 

rH 

rH 

J3 

o 

a 

rH 

05 

© 

co 

rH 

05 

X 

X 

CO 

> 



rH 


rH 


rH 


rH 




cq 

O 


co 

CO 

CO 

rH 

rH 

rH 

X 

X 

X 

X 

X 



r- 

r- 

b- 

rH 

rH 

rH 



TF 

tF 

tF 

jo 


no 

no 

no 


i> 

F- 


tF 

tF 

tF 

^f 

S3 

o 


of 

of 

of 

of 

of 

of 

of 

of 

of 

of 

of 

d 

h 

**o 

55 

no 

no 

no 

no 

no 

no 

CO 

CO 

CO 

CO 

CO 

a 

V- 

no 

no 

no 

l> 

t> 

i> 



xF 

rF 

tF 

T3 

s, 

rH 

rH 

rH 




04 

04 

04 

04 

04 

X 













-a 













O 













fal 

. 

o 

o 

o 

O 

o 

O 

O 

O 

O 

O 

O 

03 

s 

o 

o 

o 

no 

no 

no 

no 

no 

no 

no 

no 

o 

o> 

rH 

rH 

rH 




rH 

rH 

rH 

rH 

rH 


8 

no 

no 

no 

TF 

TF 

TF 


t- 

t- 

t- 

b- 



05 

05 

05 

04 

04 

04 

co 

co 

X 

X 

X 



CO 

CO 

CO 

X 

OO 

X 

CO 

CO 

CO 

CO 

CO 

H 

a 

























fa 

. 

rH 

rH 

rH 

rH 

rH 

rH 

no 

no 

no 

no 

no 


s 

o 

o 

o 

no 

no 

no 

f- 

t- 

t- 




cx 

04 

04 

04 

04 

04 

04 

rH 

rH 

rH 

rH 

rH 


Is 




rH 

rH 

rH 








no 

no 

no 

o 

d 

d 

rH 

rH 

rH 

rH 

rH 

• 





rH 

rH 

rH 

rH 

rH 

rH 

rH 

rH 

G 

OJ 













G, 












<D 

H 














OOCOOOOOOOIMININMM 
003 05COCOCDNNNNN 


X 05 O t-i 04 
rH i-H 04 04 04 


O 

0 

o 

+3> 

CP 

C5 

c3 

tM 

o 

a3 

s 

Sh 

a 

•+j 


T3 

o 

t» 

co 

a; 

?- 

Cfa 

x 

<p 

a> 

Jh 

c3 

CO 

<p 

O 

fa} 

a 

c 

o 

O) 

C5 

o3 

a> 

fa3 


co 

C 

o 

o3 

d 

s 

!-h 

<p 

-n> 

a> 

<P 

faG 

< 4-1 

O 

co 

-i-> 

co 

<p 





































372 


Studies on Acetonuria 


excretion of only very small amounts of acetone the weight was quite con¬ 
stant. There was a slightly negative nitrogen balance as based on the 
determination of urine nitrogen during the first part of the period of study, 
and a slight retention later when the diet fed was more nearly normal. 
The excretion of acetone increased from 40 mg. on the day before the experi¬ 
ment to about 1.25 gm. after 4 days on a diet which contained 10 per cent 


oi ft a mm • 

CAL 2f70 

PR lir. «.r T p crrn 

CHI5S%IOO DIET B 222 

FAT 6?.$%20l 

MARCH 1411 

'CAL Z700 

FAT $24 % 151 

CAL 2460 

PF, 11.7 % /I f«- 
CH 

FAT 63.7 %\15 

i 

2 1 

3 l 

4 

S 1 

6 17 

12 1 

4 2 

0 1 

1 2 

1 


DIET 










YN 



RATIO 

no 

tad 

no 

160 

150 

140 

130 

120 

no 

100 

50 

80 

70 

60 

50 

40 

30 

20 

10 

0 
































































































































































\ 












\ 












\ 

















































































TOTAL 4" 1 * 

ACETONE 1 

1 

0 


















Y 

V 












\ 



l 

V 














Chart 1. 


of the calories as protein, 10 per cent as carbohydrate, and 80 per cent as 
fat; this increase makes it seem probable that there were more ketogenic 
than antiketogenic compounds in the diet. When the relative amount of 
carbohydrate in the diet was increased the excretion of the acetone bodies 
diminished, but did not return to the normal values when the diet contained 
20 per cent of the calories as carbohydrate and 70 per cent as fat; oleomar¬ 
garine and olive oil were substituted for the larger part of the butter fat in 



























































R. S. Hubbard and F. R. Wright 


373 


this diet for 3 days, the 7th, Sth, and 9th of August, but the excretion of 
the acetone bodies was not measurably decreased further. However, the 
amounts of acetone excreted when this diet, which has a ketogenic balance 
of 108 per cent, was fed were not markedly different from those found on a 
less severe diet which had a ketogenic balance of 79 per cent, and were not 
very large in either case. The border-line of increased acetonuria appears 
to lie, for this case, between diets giving values of 78 and 108 per cent, 
although the value may be higher if the excretion of very small amounts of 
acetone is- regarded as important in determining when ketogenic antiketo¬ 
genic equilibrium has been established. It is noticeable, from comparing 
these two experiments on the same individual, that the excretion of acetone 
depends on the ratio between fat and carbohydrate rather than on the fat 
content of the diet. 

Detailed Diet. 

Case IV. 

July 15. 

Breakfast: Eggs, 2; Bacon, 20 gm.; Cream, 45 cc.; Butter, 15 gm.; 
Bread, 15 gm.; 10 per cent fruit, 70 gm. 

Dinner: Meat, 60 gm.; 5 per cent vegetable, 135 gm.; Potato, 30 gm.; 
Cream, 40 cc.; Cheese, 15 gm.; Bread, 15 gm.; Butter, 25 gm. 

Supper: Meat, 30 gm.; 5 per cent vegetable, 135 gm.; Bacon, 10 gm.; 
Cream, 35 cc.; Cheese, 15 gm.; 10 per cent fruit, 50 gm.; Bread, 15 gm.; 
Butter, 27 gm. 

Extras (one-third to each meal): Olive oil, 90 cc.; Lemon, 50 cc. Fore¬ 
noon—Olive oil, 30 cc.; Lemon, 15 cc. 

Summary: Carbohydrate, 63 gm.; Protein, 63 gm.; Fat, 222 gm. 

July 16. 

Breakfast: Eggs, 2; Bacon, 20 gm.; Bread, 15 gm.; Cream, 60 cc.; 
Butter, 20 gm.; 10 per cent fruit, 80 gm. 

Dinner: Meat, 60 gm.; Bread, 10 gm.; 5 per cent vegetable, 120 gm.; 
Potato, 30 gm.; Cheese, 15 gm.; Cream, 60 cc.; Butter, 30 gm.; Water¬ 
melon, 45 gm. 

Supper: Meat, 30 gm.; 5 per cent vegetable, 120 gm.; Cheese, 15 gm.; 
Butter, 25 gm.; Bacon, 10 gm.; Cream, 60 cc.; Bread, 20 gm.; Watermelon, 
45 gm. 

Extras (one-third to each meal): Olive oil, 90 cc.; Lemon, 20 cc. 

Summary: Carbohydrate, 63 gm.; Protein, 63 gm.; Fat, 222 gm. 

July 17. 

Breakfast: Eggs, 2; Bacon, 20 gm.; Bread, 15 gm.; Cream, 60 cc.; 
Butter, 20 gm.; 10 per cent fruit, 80 gm. 

Dinner: Meat, 60 gm.; Bread, 10 gm.; 5 per cent vegetable, 120 gm.; 
Potato, 30 gm.; Cheese, 15 gm.; Cream, 60 cc.; Butter, 30 gm.; Water¬ 
melon, 45 gm. 


374 


Studies on Acetonuria 


Supper: Meat, 30 gm.; 5 per cent vegetable, 120 gm.; Cheese, 15 gm.; 
Butter, 25 gm.; Bacon, 10 gm.; Cream, 60 cc.; Bread, 20 gm.; Watermelon, 
45 gm. 

Extras (one-third to each meal): Olive oil, 90 cc.; Lemon, 20 cc. 
Summary: Carbohydrate, 63 gm.; Protein, 63 gm.; Fat, 222 gm. 

July 18. 

Breakfast: Eggs, 2; Bacon, 20 gm.; Bread, 15 gm.; Cream, 60 cc.; 
Butter, 20 gm.; 10 per cent fruit, 80 gm. 

Dinner: Meat, 60 gm.; Bread, 10 gm.; 5 per cent vegetable, 120 gm.; 
Potato, 30 gm.; Cheese, 15 gm.; Cream, 60 cc.; Butter, 30 gm.; Water¬ 
melon, 45 gm. 

Supper: Meat, 30 gm.; 5 per cent vegetable, 120 gm.; Cheese, 15 gm.; 
Butter, 25 gm.; Bacon, 10 gm.; Cream, 60 cc.; Bread, 20 gm.; Watermelon, 
45 gm. 

Extras (one-third to each meal): Olive oil, 90 cc.; Lemon, 20 cc. 
Summary: Carbohydrate, 63 gm.; Protein, 63 gm.; Fat, 222 gm. 

July 19. 

Breakfast: Eggs, 2; Bacon, 30 gm.; Bread, 10 gm.; Butter, 20 gm.; 
Cream, 30 cc.; Watermelon, 60 gm. 

Dinner: Meat, 60 gm.; 5 per cent vegetable, 135 gm.; Cheese, 15 gm.; 
Butter, 34 gm.; Cream, 30 cc.; Watermelon, 60 gm. 

Supper: Meat, 30 gm.; Bacon, 30 gm.; 5 per cent vegetable, 135 gm.; 
Cream, 30 cc.; Cheese, 15 gm.; Butter, 30 gm.; Watermelon, 60 gm. 

Extras (one-third to each meal): Olive oil, SO cc., Lemon, 15 cc. 
Summary: Carbohydrate, 31.5 gm.; Protein, 63 gm.; Fat, 236 gm. 

July 20. 

Breakfast: Eggs, 2; Bacon, 30 gm.; Butter, 30 gm.; Cream, 90 cc.; 
Watermelon, 100 gm. 

Dinner: Meat, 45 gm.; Cheese, 15 gm.; 5 per cent vegetable, 180 gm.; 
Butter, 30 gm.; Cream, 60 cc.; Watermelon, 65 gm. 

Supper: Meat, 30 gm.; Bacon, 30 gm.; 5 per cent vegetable, 180 gm.; 
Cheese, 15 gm.; Butter, 27 gm.; Cream, 60 cc. 

Extras (one-third to each meal): Olive oil, 60 cc.; Lemon, 15 cc. 
Summary: Carbohydrate, 31.5 gm.; Protein, 63 gm.; Fat, 236 gm. 

July 21. 

Breakfast: Eggs, 2; Bacon, 30 gm.; Butter, 25 gm.; Cream, 60 cc.; 
Watermelon, 100 gm. 

Dinner: Meat, 45 gm.; 5 per cent vegetable, 180 gm.; Cheese, 15 gm.; 
Butter, 30 gm.; Cream, 90 cc.; Watermelon, 65 gm. 

Supper: Meat, 30 gm.; Bacon, 30 gm.; 5 per cent vegetable, 180 gm.; 
Cheese, 15 gm.; Butter, 27 gm.; Cream, 60 cc. 

Extras (one-third to each meal): Olive oil, 60 cc.; Lemon, 15 cc. 
Summary: Carbohydrate, 31.5 gm.; Protein, 63 gm.; Fat, 236 gm. 


R. S. Hubbard and F. R. Wright 


375 


July 22. 

Breakfast: Eggs, 2; Bacon, 30 gm.; 10 per cent fruit, 100 gm.; Bread, 
15 gm.; Butter, 20 gm.; Cream, 60 cc. 

Dinner: Meat, 45 gm.; 5 per cent vegetable, 90 gm.; Bread, 15 gm.; 
10 per cent fruit, 40 gm.; Butter, 30 gm.; Cheese, 15 gm.; Cream, 60 cc. 

Supper: Meat, 30 gm.; Cheese, 15 gm.; Bacon, 25 gm.; 5 per cent vege¬ 
table, 90 gm.; 10 per cent fruit, 55 gm.; Bread, 20 gm.; Butter, 25 gm.; 
Cream, 60 cc. 

Extras (one-third to each meal): Olive oil, 60 cc.; Lemon, 15 cc. 
Summary: Carbohydrate, 63 gm.; Protein, 63 gm.; Fat, 222 gm. 

July 23. 

Breakfast: Eggs, 2; Bacon, 30 gm.; 10 per cent fruit, 100 gm.; Bread, 
15 gm.; Butter, 20 gm.; Cream, 60 cc. 

Dinner: Meat, 45 gm.; 5 per cent vegetable, 90 gm.; 10 per cent fruit, 
40 gm.; Bread, 15 gm.; Cheese, 15 gm.; Butter, 30 gm.; Cream, 60 cc. 

Supper: Meat, 30 gm.; Bacon, 25 gm.; Cream, 60 cc.; 5 per cent vege¬ 
table, 90 gm.; Butter, 25 gm.; 10 per cent fruit, 55 gm.; Bread, 20 gm.; 
Cheese, 15 gm. 

Extras (one-third to each meal): Olive oil, 80 cc.; Lemon, 10 cc. 
Summary: Carbohydrate, 63 gm.; Protein, 63 gm.; Fat, 242 gm. 

July 24. 

Breakfast: Eggs, 2; Bread, 15 gm.; Butter, 20 gm.; Cream, 60 cc.; 
Bacon, 30 gm.; 10 per cent fruit, 100 gm. 

Dinner: Meat, 45 gm.; 5 per cent vegetable, 90 gm.; 10 per cent fruit, 
40 gm.; Bread, 15 gm.; Cheese, 15 gm.; Butter, 30 gm.; Cream, 60 cc. 

Supper: Meat, 30 gm.; Bacon, 25 gm.; Cream, 60 cc.; Butter, 25 gm.; 
5 per cent vegetable, 90 gm.; 10 per cent fruit, 55 gm.; Bread, 20 gm.; Cheese, 
15 gm. 

Extras (one-third to each meal): Olive oil, 40 cc.; Lemon, 10 cc. 
Summary: Carbohydrate, 63 gm.; Protein, 63 gm.; Fat, 202 gm. 

July 25. 

Breakfast: Eggs, 2; Bacon, 30 gm.; 10 per cent fruit, 100 gm.; Bread, 
15 gm.; Butter, 20 gm.; Cream, 60 cc. 

Dinner: Meat, 45 gm.; 5 per cent vegetable, 90 gm.; 10 per cent fruit, 
40 gm.; Cheese, 15 gm.; Bread, 15 gm.; Butter, 30 gm.; Cream, 60 cc. 

Supper: Meat, 30 gm.; Bacon, 25 gm.; 5 per cent vegetable, 90 gm.; 
10 per cent fruit, 55 gm.; Cheese, 15 gm.; Bread, 20 gm.; Butter, 25 gm.; 
Cream, 60 cc. 

Extras (one-third to each meal): Olive oil, 60 cc.; Lemon, 15 cc. 
Summary: Carbohydrate, 63 gm.; Protein, 63 gm.; Fat, 222 gm. 

July 26. 

Breakfast: Eggs, 2; Bacon, 30 gm.; 10 per cent fruit, 150 gm.; Bread, 
20 gm.; Butter, 30 gm.; Cream, 90 cc. 



376 


Studies on Acetonuria 


Dinner: Meat, 60 gm.; Bacon, 20 gm.; 5 per cent vegetable, 90 gm.; 
Potato, 30 gm.; Bread, 35 gm.; Butter, 25 gm.; Cream, 45 cc. 

Supper: Bacon, 40 gm.; 5 per cent vegetable, 90 gm.; Potato, 30 gm.; 
Bread, 35 gm.; Butter, 35 gm.; Cream, 45 cc. 

Extras (one-third to each meal): Olive oil, 30 cc.; Lemon, 10 cc. 
Summary: Carbohydrate, 94 gm.; Protein, 63 gm.; Fat, 208 gm. 

July 27. 

Breakfast: Eggs, 2; Bacon, 30 gm.; 10 per cent fruit, 150 gm.; Bread, 
20 gm.; Butter, 30 gm.; Cream, 90 cc. 

Dinner: Meat, 60 gm.; 5 per cent vegetable, 90 gm.; Cream, 45 cc.; 
Bacon, 20gm.; Potato, 30 gm.; Bread, 35 gm.; Butter, 30 gm. 

Supper: Bacon, 40 gm.; 5 per cent vegetable, 90 gm.; Potato, 30 gm.; 
Cream, 45 cc.; Bread, 35 gm.; Butter, 30 gm. 

Extras (one-third to each meal): Olive oil, 30 cc.; Lemon, 10 cc. 
Summary: Carbohydrate, 94 gm.; Protein, 63 gm.; Fat, 208 gm. 

July 28. 

Breakfast: Eggs, 2; Bacon, 30 gm.; 10 per cent fruit, 150 gm.; Bread, 20 
gm.; Butter, 30 gm.; Cream, 90 cc. 

Dinner: Meat, 60 gm.; Bacon, 20 gm.; 5 per cent vegetable, 90 gm.; 
Potato, 30 gm.; Bread, 35 gm.; Butter, 30 gm.; Cream, 45 cc. 

Supper: Bacon, 40 gm.; 5 per cent vegetable, 90 gm.; Potato, 30 gm.; 
Bread, 35 gm.; Butter, 30 gm.; Cream, 45 cc. 

Extras (one-third to each meal): Olive oil, 30 cc.; Lemon, 10 cc. 
Summary: Carbohydrate, 94 gm.; Protein, 63 gm.; Fat, 208 gm. 

July 29. 

Breakfast: Eggs, 2; Bacon, 30 gm.; 10 per cent fruit, 150 gm.; Bread, 
20 gm.; Butter, 30 gm.; Cream, 90 cc. 

Dinner: Meat, 60 gm.; Bacon, 20 gm.; 5 per cent vegetable, 90 gm.; 
Potato, 30 gm.; Bread, 35 gm.; Cream, 45 cc.; Butter, 30 cc. 

Supper: Bacon, 40 gm.; 5 per cent vegetable, 90 gm.; Potato, 30 gm.; 
Bread, 35 gm.; Butter, 30 gm.; Cream, 45 cc. 

Extras (one-third to each meal): Olive oil, 30 cc.; Lemon, 10 cc. 
.Summary: Carbohydrate, 94 gm.; Protein, 63 gm.; Fat, 208 gm. 

July 30. 

Breakfast: Eggs, 2; Bacon, 30 gm.; 10 per cent fruit, 100 gm.; Bread, 
15 gm.; Butter, 20 gm.; Cream, 60 cc. 

Dinner: Meat, 45 gm.; Cheese, 15 gm.; 5 per cent vegetable, 90 gm.; 
10 per cent fruit, 40 gm.; Bread, 15 gm.; Butter, 30 gm.; Cream, 60 cc. 

Supper: Meat, 30 gm.; Bacon, 25 gm.; 5 per cent vegetable, 90 gm.; 
Cream, 60 cc.; Cheese, 15 gm.; 10 per cent fruit, 55 gm.; Bread, 20 gm.; 
Butter, 25 gm. 

Extras (one-third to each meal): Olive oil, 60 cc.; Lemon, 15 cc. 
Summary: Carbohydrate, 63 gm.; Protein, 63 gm.; Fat, 222 gm. 


R. S. Hubbard and F. R. Wright 


377 


% July 31. 

Breakfast: Eggs, 2; Bacon, 30 gm.; 10 per cent fruit, 100 gm.; Bread, 
15 gm.; Butter, 20 gm.; Cream, 60 cc. 

Dinner: Meat, 45 gm.; 5 per cent vegetable, 90 gm.; Cheese, 15 gm.; 
10 per cent fruit, 40 gm.; Bread, 15 gm.; Cream, 60 cc.; Butter, 30 gm. 

Supper: Duck, 40 gm.; Bacon, 25 gm.; 5 per cent vegetable, 90 gm.; 
Cheese, 15 gm.; Bread, 20 gm.; Butter, 22 gm.; 10 per cent fruit, 55 gm.; 
Cream, 60 cc. 

Extras (one-third to each meal): Olive oil, 60 cc.; Lemon, 15 cc. 
Summary: Carbohydrate, 63 gm.; Protein, 63 gm.; Fat, 222 gm. 

Aug. 1. 

Breakfast: Eggs, 2; Bacon, 30 gm.; 10 per cent fruit, 100 gm.; Bread, 
15 gm.; Butter, 20 gm.; Cream, 60 cc. 

Dinner: Meat, 45 gm.; Cheese, 15 gm.; 5 per cent vegetable, 90 gm.; 
10 per cent fruit, 40 gm.; Bread, 15 gm.; Butter, 30 gm.; Cream, 60 cc. 

Supper: Meat, 30 gm.; Cheese, 15 gm.; Bacon, 25 gm.; 5 per cent vege¬ 
table, 90 gm.; 10 per cent fruit, 55 gm.; Bread, 20 gm.; Butter, 25 gm.; 
Cream, 60 cc. 

Extras (one-third to each meal): Olive oil, 60 cc.; Lemon, 15 cc. 
Summary: Carbohydrate, 63 gm.; Protein, 63 gm.; Fat, 222 gm. 

Aug. 2. 

Breakfast: Egg, 1; Bacon, 30 gm.; Oats, 10 gm.; 10 per cent fruit, 110 
gm.; Bread, 30 gm.; Butter, 30 gm.; Milk, 60 cc.; Cream, 90 cc. 

Dinner: Meat, 30 gm.; Potato, 30 gm.; 10 per cent fruit, 100 gm.; 5 per 
cent vegetable, 60 gm.; Bread, 30 gm.; Bacon, 25 gm.; Butter, 30 gm.; 
Cream, 90 cc.; Milk, 60 cc. 

Supper: Egg, 1; Bacon, 30 gm.; 10 per cent fruit, 100 gm.; 5 per cent 
vegetable, 60 gm.; Bread, 30 gm.; Potato, 30 gm.; Butter, 30 gm.; Cream, 
90 cc.; Milk, 30 cc. 

Summary: Carbohydrate, 125 gm.; Protein, 63 gm.; Fat, 194 gm. 

Aug. 3. 

Breakfast: Egg, 1; Bacon, 30 gm.; Oats, 10 gm.; 10 per cent fruit, 110 
gm.; Bread, 30 gm.; Butter, 30 gm.; Milk, 60 cc.; Cream, 90 cc. 

Dinner: Meat, 30 gm.; Bacon, 25 gm.; 5 per cent vegetable, 60 gm.; 
Potato, 30 gm.; 10 per cent fruit, 100 gm.; Bread, 30 gm.; Butter, 30 gm.; 
Cream, 90 cc.; Milk, 60 cc. 

Supper: Egg, 1; Bacon, 30 gm.; 5 per cent vegetable, 60 gm.; Potato, 
30 gm.; 10 per cent fruit, 100 gm.; Bread, 30 gm.; Butter, 30 gm.; Cream, 90 
cc.; Milk, 30 cc. 

Summary: Carbohydrate, 125 gm.; Protein, 63 gm.; Fat, 194 gm. 


378 


Studies on Acetonuria 


Aug. 4. 

Breakfast: Egg, 1; Bacon, 30 gm.; Oats, 10 gm.; 10 per cent fruit, 110 
gm.; Bread, 30 gm.; Butter, 30 gm.; Milk, 60 cc.; Cream, 90 cc. 

Dinner: Meat, 30 gm.; Bacon, 25 gm.; 5 per cent vegetable, 60 gm.; 
Potato, 30 gm.; 10 per cent fruit, 100 gm.; Bread, 30 gm.; Butter, 30 gm.; 
Cream, 90 cc.; Milk, 60 cc. 

Supper: Egg, 1; Bacon, 30 gm.; Potato, 30 gm.; 5 per cent vegetable, 
60 gm.; 10 per cent fruit, 100 gm.; Bread, 30 gm.; Butter, 30 gm.; Cream, 
90 cc.; Milk, 30 cc. 

Summary: Carbohydrate, 125 gm.; Protein, 63 gm.; Fat, 194 gm. 

Aug. 5. 

Breakfast: Egg, 1; Bacon, 30 gm.; Oats, 10 gm.; 10 per cent fruit, 100 
gm.; Bread, 30 gm.; Butter, 30 gm.; Milk, 60 cc.; Cream, 90 cc. 

Dinner: Meat, 30 gm.; Bacon, 25 gm.; 5 per cent vegetable, 60 gm.; 
Potato, 30 gm.; 10 per cent fruit, 100 gm.; Bread, 30 gm.; Butter, 30 gm.; 
Cream, 90 cc.; Milk, 60 cc. 

Supper: Egg, 1; Bacon, 30 gm.; 5 per cent vegetable, 60 gm.; Potato, 
30 gm.; 10 per cent fruit, 100 gm.; Bread, 30 gm.; Butter, 30 gm.; Milk, 30 
cc.; Cream, 90 cc. 

Summary: Carbohydrate, 125 gm.; Protein, 63 gm.; Fat, 194 gm. 

Aug. 6. 

Breakfast: Egg, 1; Bacon, 30 gm.; Oats, 10 gm.; 10 per cent fruit, 110 
gm.; Bread, 30 gm.; Butter, 30 gm.; Milk, 60 cc.; Cream, 90 cc. 

Dinner: Meat, 30 gm.; Bacon, 25 gm.; 5 per cent vegetable, 60 gm.; 
Potato, 30 gm.; 10 per cent fruit, 100 gm.; Bread, 30 gm.; Butter, 30 gm.; 
Cream, 90 cc.; Milk, 60 cc. 

Supper: Egg, 1; Bacon, 30 gm.; 5 per cent vegetable, 60 gm.; Potato, 
30 gm.; 10 per cent fruit, 100 gm.; Bread, 30 gm.; Butter, 30 gm.; Cream, 
90 cc.; Milk, 30 cc. 

Summary: Carbohydrate, 125 gm.; Protein, 63 gm.; Fat, 194 gm. 

Aug. 7. 

Breakfast: Egg, 1; Egg white, 1; Bacon, 30 gm.; Oleo, 30 gm.; Oats, 
10 gm.; 10 per cent fruit, 200 gm.; Bread, 30 gm.; Milk, 60 cc.; Cream, 30 cc. 

Dinner: Meat, 30 gm.; Bacon, 25 gm.; 5 per cent vegetable, 150 gm.; 
Potato, 30 gm.; 10 per cent fruit, 50 gm.; Bread, 30 gm.; Oleo, 30 gm.; 
Cream, 20 cc.; Milk, 60 cc. 

Supper: Egg, 1; Bacon, 30 gm.; 5 per cent vegetable, 150 gm.; Potato, 
30 gm.; 10 per cent fruit, 50 gm.; Bread, 30 gm.; Oleo, 30 gm.; Milk, 30 cc.; 
Cream, 30 cc. 

Extras (one-third to each meal): Olive oil, 30 cc.; Lemon, 10 cc. 
Summary: Carbohydrate, 125 gm.; Protein, 63 gm.; Fat, 194 gm. 


R. S. Hubbard and F. R. Wright 


379 


Aug. 8. 

Breakfast: Egg, 1; Egg white, 1; Bacon, 30 gm.; Oats, 10 gm.; 10 per 
cent fruit, 200 gm.; Oleo, 30 gm.; Bread, 30 gm.; Cream, 30 cc.; Milk, 60 cc. 

Dinner: Meat, 30 gm.; Bacon, 25 gm.; Potatoes, 30 gm.; 5 per cent 
vegetable, 150 gm.; 10 per cent fruit, 50 gm.; Bread, 30 gm.; Oleo, 30 gm.; 
Cream, 30 cc.; Milk, 60 cc. 

Supper: Egg, 1; Bacon, 30 gm.; 10 per cent fruit, 50 gm.; 5 per cent 
vegetable, 150 gm.; Oleo, 30 gm.; Potato, 30 gm.; Bread, 30 gm.; Milk, 30 
cc.; Cream, 30 cc. 

Extras (one-third to each meal): Olive oil, 30 cc.; Lemon, 10 cc. 

Summary: Carbohydrate, 125 gm.; Protein, 63 gm.; Fat, 194 gm. 

Aug. 9. 

Breakfast: Egg, 1; Egg white, 1; Bacon, 30 gm.; Oats, 10 gm.; 10 per 
cent fruit, 200 gm.; Bread, 30 gm.; Oleo, 30 gm.; Cream, 30 cc.; Milk, 
60 cc. 

Dinner: Meat, 30 gm.; Bacon, 25 gm.; Potato, 30 gm.; 5 per cent vege¬ 
table, 150 gm.; 10 per cent fruit, 50 gm.; Bread, 30 gm.; Oleo, 30 gm.; Milk, 
60 cc.; Cream, 30 cc. 

Supper: Egg, 1; Bacon, 30 gm.; 10 per cent fruit, 50 gm.; 5 per cent 
vegetable, 150 gm.; Oleo, 30 gm.; Bread, 30 gm.; Potato, 30 gm.; Milk, 30 
cc.; Cream, 30 cc. 

Extras (one-third to each meal): Olive oil, 30 cc.; Lemon, 10 cc. 

Summary: Carbohydrate, 125 gm.; Protein, 63 gm.; Fat, 194 gm. 

Aug. 10. 

Breakfast: Eggs, 2; Bacon, 30 gm.; 10 per cent fruit, 150 gm.; Bread, 20 
gm.; Butter, 30 gm.; Cream, 90 cc. 

Dinner: Meat, 60 gm.; Potato, 30 gm.; Bacon, 20 gm.; 5 per cent vege¬ 
table, 90 gm.; Bread, 35 gm.; Butter, 25 gm.; Cream, 45 cc. 

Supper: Bacon, 40 gm.; Potato, 30 gm.; 5 per cent vegetable, 90 gm.; 
Bread, 35 gm.; Butter, 35 gm.; Cream, 45 cc. 

Extras (one-third to each meal): Olive oil, 30 cc.; Lemon, 10 cc. 

Summary: Carbohydrate, 94 gm.; Protein, 63 gm.; Fat, 208 gm. 

Aug. 11. 

Breakfast: Eggs, 2; Bacon, 30 gm.; 10 per cent fruit, 150 gm.; Bread, 20 
gm.; Butter, 30 gm.; Cream, 90 cc. 

Dinner: Meat, 60 gm.; Potato, 30 gm.; Bacon, 20 gm.; 5 per cent vege¬ 
table, 150 gm.; Bread, 35 gm.; Butter, 25 gm.; Cream, 45 cc. 

Supper: Bacon, 40 gm.; Potato, 30 gm.; 5 per cent vegetable, 90 gm.; 
Bread, 35 gm.; Butter, 35 gm.; Cream, 45 cc. 

Extras (one-third to each meal): Olive oil, 30 cc.; Lemon, 10 cc. 

Summary: Carbohydrate, 94 gm.; Protein, 63 gm.; Fat, 208 gm. 

20 per cent cream was used. All food was weighed after cooking. 


TABLE II. 

Case IV. 


380 


Studies on Acetonuria 





r- 

0 

CD 

CO 

00 









CD 

00 

CD 

Cl 

CO 


rH 

r- 




rH 

CD 

rf 

CO 

Cl 

to 

r- 

h- 

CO 

CD 

CD 

05 

CO 

CO 

to 

CO 

CO 


rH 

CD 

co 



S 

0 

r-H 

r-H 

to 

0 

CO 

CO 

CO 

0 

0 

H 

CO 

to 

t> 


CO 

CO 

to 

05 

to 

CO 


X O 

Ca> 

d 

d 

d 

0 

0 

t-H 

d 

to 


CO 

CO 

i-H 

0 

0 

d 

0 

0 

0 

O 

d 

d 


O cS 
























'S.g 

£>> 

1 

8 

0 

0 

r-H 

10 

0 











to 

CD 

Ttt 

05 

co 


00 

CO 


03 . ^ 

•*-<« 

CO 

CD 

i-H 

to 

CO 

CO 

CO 

CO 

CD 

T)H 


Tt< 

Tti 

'C 

d 

to 

CO 

0 

O 

0 

00 




CO 

CO 

t- 

CO 

r- 

to 

GO 

CD 

CD 

CD 

I- 

CD 

05 

to 

CO 

Th 

r- 

rH 

CD 

r- 



1 



l-H 

l-H 

CO 

CO 

CO 

CO 

CO 

i-H 







rH 






CD 

0 

'C 

t- 

CO 

r- 







CO 

CO 

CD 

CO 

CO 

t- 

GO 


to 




co 

10 

CO 

00 

I- 

05 

3 

I- 

0 

to 

CO 

0 

O 

CD 

05 

05 

CO 

rH 

O 

05 

05 


+ . 
©.2 

s 

0 

CO 

CO 

to 


00 

rH 

l> 

'C 

0 


CD 

to 

CO 

CO 

CO 

05 

CD 

to 



<& 

d 

0 

0 

0 

d 

0 

i-H 

CO 

rH 

rH 

rH 

d 

O 

0 

0 

O 

0 

O 

d 

0 

d 


£ 























. 

0 g 

O 






















o 

H> 2 

O 






















a 


O 

to 

i-H 

CO 









co 

to 

0 

CO 

CO 

05 


0 

t-H 

0 

Tl 

£3 


e. 

>~H 

Tt* 

0 

Tti 

r-H 

r- 

to 

0 

r- 

'C 

co 

to 

to 

T|H 

i-H 

r- 

CO 

CO 

f- 

00 


CD 



. 


CO 

CO 

00 

CD 

rH 

CO 

to 

to 

1- 

CO 

05 

t- 

t- 




rH 

t- 

CD 

•'C 



ca> 

s 






t-H 

r-H 

i-H 

t-H 

rH 

rH 







i-H 





£ 


CO 

0 

r- 

r-H 

O 

to 








0 


to 

to 

0 

CO 

Tfl 

CO 



1- 

uO 

01 

co 

O 

r- 

0 

co 

0 

0 


05 


0 

CD 

00 

0 

0 

0 

rH 

CO 


a 

s 

CO 

T*H 

Tti 

CD 

1> 

05 

CO 

CD 

00 

co 

0 

i-H 

i-H 

05 

t- 

CD 

0 

CD 

0 

to 

CD 


5 » 

O 

O 

0 

O 

O 

O 

i-H 

t-H 

1—H 

l-H 

rH 

rH 

rH 

O 

O 

0 

0 

O 

d 

0 

O 

















TJH 

O 


0 

r- 



'00 


"eS . 


CO 

00 

05 


r-H 

to 

CD 

00 

co 

CD 


05 

t- 

i— 

05 

CO 

CO 

r- 


t-H 

r*n 


oZ 

s 

r-H 

CO 

CO 

d 

CO 

CO 

CO 

co 

CO 

i-H 


O 

0 

05 

05 

0 

05 

05 

d 

0 

O 


H 


i-H 

r-H 

r-H 

i-H 

r-H 

l-H 

i-H 

i-H 

rH 

i-H 


i-H 

i-H 



rH 



rH 

t-H 



A © 


0 

0 

to 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

O 

O 

0 

»o 

O 

0 

to 

O 



CD 


r- 

co 

0 

00 

0 

GO 

0 


co 

rfi 

co 

GO 


CO 

to 

GO 

CO 

CO 

8 



10 

CD 

CD 

r- 

ic 

r- 

CO 

CO 

l-H 

00 

00 

t- 

00 


GO 

05 

t- 

t- 

00 

05 










rH 

rH 

i-H 












rH 

1 

0 

0 


s' 


O 

CD 


CO 

co 

i-H 

CO 

i-H 

C 5 


r- 

to 

CO 

O 

CD 

CO 

O 

t-H 

0 


3 -S 

s 


CO 

CO 


CO 

CO 

CO 

CO 

co 

CO 


CO 

CO 

■'tl 

Tti 

CO 



xh 



rC 


00 

00 

00 

00 



CO 

0 




0 


GO 



CO 


0 


CO 

b£l 

c 5 > 

cO 

CD 

CD 

CD 

. 


CD 

CD 

to 



to 


d 



CD 


CD 


CD 

£ 

t- 

t- 

t- 

t- 




1> 

r- 



l- 





t- 


t- 







CD 

CD 

CD 

CD 

CO 

CO 

CO 

co 

Tf 

0 

CO 

0 

O 

O 

0 

CO 

CO 

Cl 

00 

00 


1 



O 

O 

O 

O 

0 

0 

0 

0 

00 

CO 

0 

0 

O 

O 

0 

0 

0 

0 

05 

05 


J 2 CO 

15 2 


<>-• 

»o 

to 

to 

to 

to 

to 

to 

to 

CD 

CO 

to 

to 

to 

to 

to 

to 

to 

to 




0'«H 



CO 

co’ 

cT 

co” 

co” 

of 

of 

of 

cf 

co” 

co” 

co” 

co” 

co” 

co” 

co” 

•s 

CO 

co” 

co” 

co” 


g 5 

n 










to 

t- 












•4—■ 


O- 





















c 3 

u 

** 


0 

0 

0 

0 

to 

to 

to 

0 

C 5 

0 

0 

to 

to 

to 

to 

0 

0 

0 

0 

0 


T 3 

>> 



r-H 

i-H 

rH 

i-H 




i-H 


i-H 

rH 

i-H 

i-H 

rH 

i-H 

rH 

rH 

l-H 

CO 

CO 


rfl 

0 
























t -1 

s 

c^‘ 

CO 

CO 

CO 

CO 

co 

CO 

CO 

CO 

CO 

CO 

CO 


t+i 

't+i 


CO 

CO 

CO 

to 

to 


c 3 


CD 

CD 

CD 

CD 

CO 

CO 

CO 

CD 

CD 

CD 

CD 

05 

05 

05 

05 

CD 

CD 

CD 

CO 

CO 


O 





















rH 

rH 

0 > 


£ 

$ 

V 

0- 










to 













{s 


O 

O 

O 

O 

to 

to 

to 

O 

i-H 

00 

O 

to 

to 

to 

to 

0 

O 

O 

0 

0 

R 

Fat. 

a 


00 

00 

00 

00 

00 

00 

00 

00 

00 


00 


15 - 

1 > 

t- 

00 

00 

00 

1- 





CO 

CO 

CO 

CO 

CD 

CD 

CD 

CO 

CO 

CO 

CO 

00 

00 

00 

00 

CO 

CO 

CO 


'Cl 



s' 

c— 

CO 

CO 

CO 

CO 

CO 

CO 

CO 

CO 

'C 

0 

CO 

0 

0 

0 

0 

Cl 

CO 

CO 

05 

05 



C* 


CO 

CO 

CO 

CO 

CO 

CO 

CO 

CO 

CO 

CO 

CO 

co 

CO 

CO 

CO 

CO 

CO 

CO 

t-H 

"rH 



<» 

c- 









to 

l> 















0 

0 

0 

0 

0 

0 

0 

0 

05 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 


*53 

ft 


t-H 

i-H 

1—1 

t-H 

i-H 

i-H 

rH 

i-H 


t-H 

t-H 

i-H 


^H 

rH 

t-H 

i-H 

1 —H 

i-H 

i-H 


0 

H 
























Ph 

S 

<N- 

CO 

CO 

CO 

CO 

CO 

CO 

CO 

CO 

CO 

CO 

CO 

co 

CO 

CO 

CO 

CO 

CO 

CO 

co 

CO 




CD 

CD 

CD 

CD 

CD 

CD 

CD 

CD 

CD 

CD 

CD 

CD 

CD 

CD 

CD 

0 

CD 

CD 

CD 

0 



IO 

CD 


00 

O 

O 

rH 

CO 

CO 


to 

CD 

r- 

00 

05 

O 

i-H 

rH 

CO 

CO 


6 


r-H 

i-H 

l-H 

r-H 

r-H 

CO 

CO 

CO 

CO 

CO 

CO 

CO 

co 

CO 

CO 

CO 

CO 


-h 

c3 

Q 

Oi 

1 -^ 

V# 











N# 





bi) 






D 















v# 



v» 





•“5 

















< 








































R. S. Hubbard and F. R. Wright 


381 


c© 


rf 

CM 

oo 

CM 

r- 

oo 

03 

CM 

03 

03 

no 


o 

00 

CM 

CO 

O 

o 

CO 

rH 

rH 

CM 

© 

o 

d 

o 

d 

d 

o 

o 

03 

CO 

00 

oo 

CM 

CO 

CO 

co 

CM 

d 

rH 

d 

CO 

rH 

CO 

co 

CO 


rH 

rH 

Th 

CM 

rH 

co 

Th 

CO 

o 

CM 

Tfl 

o 


03 

CM 

00 

oo 

CO 

C3 

CO 

CO 

CM 

CO 

CM 

© 

rH 

rH 

rH 

rH 

CM 

o 

o 

o 

d 

d 

o 

o 

O 

rH 

no 

o 

O 

o 

o 

i> 

CO 

d 

rh 

o 

d 

CO 

d 

CO 

O 

CO 

CO 

rH 

CM 

CM 

CM 

rH 

CM 

© 


rt< 

CM 

CM 

00 

rt< 

O 

CO 


CO 

rH 

no 

00 

r- 


ro 


no 

no 


rh 

no 

CO 

d 


O 

d 

o 

o 

O 

d 

00 


o 

no 

CM 



CO 

co 



rH 

CM 

o 

1> 

CM 

rH 

03 


oo 

03 

03 

o 

d 






rH 

rH 


o 

o 

o 

o 

o 

o 

o 

o 

o 

CM 

o 

no 

CO 

o 

o 

CO 

03 

00 

oo 

00 

00 

00 

00 

CO 


rH 

o 

o 

o 

CM 




rt< 

rfl 



"'f 



rH 

00 



oo 



00 

CO 

no 

no 


no 



no 


I>- 






r- 

oo 

00 

oo 

oo 

00 

00 

o 


03 

03 

03 

03 

03 

03 

o 


rf 

rh 

rf4 

•'f 

rt< 

Tf 

no 

no 

CM 

of 

CM 

cm" 

cm" 

cm" 

cm" 

cm" 

O 

o 

O 

o 

o 

o 

no 

no 

CM 

OJ 

CM 

CM 

CM 

CM 

rH 

rH 

nc 

no 

no 

no 

no 

no 


rH 

05 

CM 

OJ 

CM 

CM 

CM 

03 

03 

rH 

rH 

H 

rH 

rH 

rH 



o 

o 

o 

o 

o 

o 

no 

no 

I> 

b- 



r- 




rH 

Tf 

3 

rt< 


% 

00 

00 

03 

03 

03 

03 

o 

o 

t— H 

rH 

rH 

rH 

rH 

rH 

CM 

CM 

o 

o 

o 

o 

o 

o 

o 

o 

rH 

rH 

H 

rH 

rH 

rH 

rH 

rH 

CO 

CO 

CO 

CO 

CO 

CO 

CO 

CO 

CO 

CO 

CO 

CO 

co 

co 

CO 

co 

rO 

CO 

w 

00 

03 

o 

rH 

CM 






rH 

tH 

rH 

ti 


V* 


s« 

V# 



o 




s* 




< 









03 

Ch 

X 

03 

03 

X! 

- 4-3 

a 

o 

A 

£ 

fl 

O 

a 

■4-3 

o 

03 

• *—a 

-Q 

to 

03 

-£3 

H 


o 

c 

o 

-4-3 

03 

C3 

(3 

«4-H 

O 

to 


S-, 

03 

-4-3 


© 

03 

rH 

03 

a 

t/2 


Q3 

£ 

?H 

a 

Oh 

• rH 
(h 

X 

03 

03 

© 

03 

* 

?-H 

c3 

-H 

03 

O 

03 

«-l-l 

• rH 

T5 

O 

T? 

03 

CQ 

0 

03 

03 

0 

0 

O 

0 

-M 

03 

03 

03 

-O 

<3 

-4-3 

03 

-O 

to 

c3 

-H5 

03 


a 

o 

c3 


03 

tn 

0 

o 

03 

-CJ 


-+3> 

-4-= 

c3 

03 

c3 

£ 

a 


Jh 

03 

03 

•+35 

a 

03 

s-i 

© 

0 


<-i-l 

«+-i 

s-l 

O 

03 

co 

a 

03 

~4+> 


C!$ 

Tt 

tc 

£ 

03 

-+35 


s 

03 


rH 


a 





















382 


Studies on Acetonuria 







Chart 2 





























































































JULY AUGUST 


R. S. Hubbard and F. R. Wright 


383 



Chart 2A 





























































































TABLEAU 

Case II. 


384 


Studies on Acetonuria 






© 

CO 

•4 

o 


CO 

© 

© 

rH 

© 

X 


© 


X 

X 





X 

© 

X 






s 

rH 


1—1 

X 

© 

CO 

CO 

X 


© 




X 




CO 

X 

rH 



• 

g 

o 

o 

rH 


1—1 

© 

rH 

rH 

X 

rH 


© 

© 

© 

© 

© 




© 

© 

© 



>3 

X O 

<a> 

d 

o 

o 

o 


d 

© 

d 

© 

© 

© 


© 

© 

© 

© 

© 




d 

© 

© 



o 




























g 


























£>> 

o 

© 

t> 

ic 

rH 


rH 


rH 

© 

© 

CO 


© 

1> 

© 


X 




© 

X 

© 




o 






















CO 




oa. S 


d 

o 

CO 

1C 


© 

d 

CO 

b- 

CO 

X 


X 


CO 

d 

CO 




CO 

rH 



ja 

t 





rH 


CO 

rH 

















b~ 

CO 

b- 

CO 


t-H 

4* 

cD 

© 

© 

X 




X 

© 

© 





X 

"4 





o 

rH 

CO 



b- 

4 

X 

© 

t-H 

X 


© 

© 

© 

© 

© 




CO 


t-H 




g 

o 

o 

O 

r-H 


t-H 

rH 

rH 

rH 

CO 

rH 


rH 

© 

© 

O 

© 




© 

© 

© 



+ CJ 

C5> 

o 

d 

o 

d 


© 

© 

© 

© 

© 

© 


© 

© 

o 

© 

© 




© 

© 

© 



o *rt 



























fi o 



























o o 



























-H $3 

o 

CO 

00 


CD 


1C 

© 

X 

© 

© 

© 


X 

rH 

CO 

© 

rH 




b- 

CO 

© 



o T5 


• 


















CO 




<1 

■*-1 

d 

o 

CO 

CD 


© 

© 

rH 

© 

© 

CO 



d 

© 

© 

d 




rH 

rH 









rH 


t-H 

rH 













6 

C5 



1 

















4 










© 

o 

CO 

cp 


© 

X 

© 

X 

4 

b- 


© 


CO 

o 

© 




© 

© 

rH 




© 

ic 

CO 



"4 

© 

4 

© 

X 

© 


a) 

X 

© 

CO 

CO 




© 

© 

X 

P 


"4 

W 

*- 

CO 

(M 

<M 

co 


CO 

X 

CO 

X 

X 

X 


CO 

CO 

rH 

CO 




CO 

CO 

CO 




O 

o 

O 

o 


© 

© 

© 

© 

© 

© 


© 

© 

© 

© 

© 




© 

© 

© 











© 



b^ 

b*. 



© 

© 












S 







rH 



© 

© 



rH 

© 











H 

C5> 







© 



© 





© 











. 

z 

x 

Cl 

CD 

rH 


rH 

© 

© 

© 

t-H 



X 

© 



© 




© 

© 

© 





© 

Cl 

CO 

CD 


© 

rH 

b- 

§ 

© 

© 


© 

GO 

CO 

X 





X 

© 

b^ 



o 

o 

CO 

CO 

CO 

CO 


X 

X 

CO 

CO 

CO 


CO 

CO 

CO 

CO 

Cl 




CO 

CO 

CO 



<! 






















. o' 

m 



ic 





© 



© 











© 

© 



4). 2 

o 

Cl 

b- 

CO 


CO 

X 

X 

4 

© 

X 



00 

b- 


© 




X 

"4 1 

"4 



«-s 

c3 

a 

CD 

»c 

ic 

ic 


© 

© 

© 

© 

© 

© 


© 

© 

© 

© 

© 




© 

© 

© 





o 

o 

o 

o 


© 

© 

© 

© 

© 

© 


© 

© 

© 

© 

© 




© 

© 

© 



^i, 0) 
>1 


b~ 

CO 

CD 

"4 


© 

© 

X 

b^ 

© 

X 



© 

©) 

CO 

© 




© 

o 

X 





I> 


t-H 


t> 

t-H 

X 

© 

4 

X 


CO 

© 

X 

CO 

T)H 




X 

X 

X 



<o 


























K- ^ 


rH 

rH 

rH 

CO 


rH 

CO 

rH 

rH 

rH 

rH 


rH 

rH 

rH 

rH 

rH 




rH 

rH 

rH 


■+3 

T& 



© 

o 

J> 


CO 

© 

© 

o 

rH 

X 

1> 


TJH 

X 

CO 

CO 


© 

CO 

CO 

CO 

CO 


1 



on 

00 

b- 


b- 

b- 

b- 



© 

© 

© 

© 

© 

© 

© 


© 

© 

© 

© 

© 




CD 

CD 

CD 


© 

© 

© 

© 

© 

© 

© 

© 

© 

© 

© 

© 


© 

© 

© 

© 

© 






00 

00 

00 

00 

X 

X 

X 

X 

X 

X 

© 

© 

© 

© 

© 

© 

© 

CO 

© 

© 

© 

© 







b~ 

b- 

b'- 


i> 



t- 

b- 

CO 

CO 

CO 

CO 

CO 

rH 

t-H 

CO 

rH 

rH 

rH 

rH 



CO 
c3 2 


c^. 

"4 

"4 

"4 

"4 

"4 

4 

4 

4 

4 

rti 




TtH 


X 

X 

rH 

X 

X 

X 

X 



O’C 



H 

t-H 

r-T 

rH 

r-H 

rH 

rH 

rH 

rH 

rH 

rH 

rH 

t-T 

rH 

rH 

rH 

rH 

rH 

rH 

rH 

rH 

rH 



<D* 

H> 

Pi 

£ 

o 

p. 

c~ 

© 

d 

9.5 

9.5 

ic 

Cl 

© 

© 

9.5 

9.5 

9.5 

© 

© 

9.5 

d 

TtH 

© 

TJH 

© 

© 

TtH 

© 

© 

© 


© 

© 

© 

© 
















rH 

rH 

rH 

rH 

rH 

rH 

rH 


t-H 

t-H 

rH 

rH 



>> 

a 


























A 

O 



























Pi 

gm. 

<b~ 

CD 

CD 

CD 

CD 

© 

© 

© 

© 

© 

© 



b- 


b^ 

X 

X 

© 

X 

X 

X 

X 



c3 

o 


CO 

CO 

CO 

CO 

CO 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

© 

© 

© 

© 

© 

© 

© 




-*o 



























a 












© 

© 

© 

© 

© 

rH 

rH 


r-H 

rH 

rH 

rH 

<D 




&-• 




















• 

• 

• 




V- 


rH 

t-H 

t-H 

t-H 

rH 

rH 

rH 

rH 

rH 

rH 

© 

© 

© 

© 

© 

© 

© 


© 

© 

© 

© 

P 


Fat. 

CD 


00 

GO 

oo 

X 

X 

X 

X 

X 

X 

X 







b» 


b^ 


b^ 

b~ 





CO 

CO 

CO 

CO 

X 

X 

X 

X 

X 

X 

© 

© 

© 

© 

© 

X 

X 

o 

X 

X 

X 

X 




S 

a> 

c— 

CO 

CO 

CO 

CO 

X 

X 

X 

X 

X 

X 


CO 

CO 

CO 

CO 

© 

© 

© 

© 

© 

© 

© 





t-H 

t-H 

t-H 

t-H 

rH 

rH 

rH 

t-H 

rH 

t-H 

rH 

rH 

rH 

rH 

rH 

rH 


rH 

rH 

rH 

rH 




8 

§ 

e— 

© 

1C 

IC 

ic 

© 

© 

© 

© 

© 

© 















£ 

$> 


Cl 

Cl 

Cl 

Cl 

© 

© 

© 

© 

© 

© 

© 

o 

© 

© 

© 

© 

© 


o 

© 

© 

© 















rH 

rH 

rH 

rH 

rH 

rH 

t-H 


rH 

rH 

rH 

rH 



<U 

a 


























O 

P< 



























P 

8 

CS 

o- 

© 

CD 

CD 

CD 

© 

© 

© 

© 

© 

© 

© 

© 

© 

© 

© 

© 

© 

X 

© 

© 

© 

© 





CO 

CO 

CO 

CO 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

rb 

"41 

rH 

"4 

"4i 

"4 

"4 





CD 

r- 

00 

Cl 

© 

rH 

CO 

X 

4 

© 

© 

t- 

X 

t-H 

CO 

X 

Tb 

© 

© 


X 

© 

© 


C3 


t-H 

t-H 

rH 

t-H 

CO 

CO 

CO 

CO 

CO 

CO 

CO 

CO 

CO 



rH 


c3 

Q 

Ot, 

•*-S 

rO 













C 

gj 














O 



v* 

v! 

s» 

N# 

d 

s# 

** 


V# 



d 

d 

5 



v 


V* 






fP 













<5 











Results of determinations on the acetone bodies are expressed in terms of acetone. 









































385 


R. S. Hubbard and F. R. Wright 

Case II was a woman, Miss A. G., aged 47 years, whose height was 5 ft. 
2 in. and who weighed 137 lbs. Her basal metabolism measured 1,335 
calories per day. Like all of the pathological cases included in the series, 
she was a severe chronic arthritic of long standing. She made every effort 
to cooperate, but after a little more than a week she found it difficult to 
take the basal diet, and it was accordingly modified as shown in Table III 
and Chart 3. The diet fed during the first part of the experiment did not 
contain enough food to maintain the weight of the patient, and there was 
a progressive loss during the first 2 weeks. In four out of five determina- 


DIET A BBS 

CAL 1478 

ch lwST olfT&nmD 

FAT8J 

FEBRUARY 

'CAL 1426 [ 

FAT7W J2.6 

MAPICH 

CAL 1816 

PR I0%45.49"- 
CH JS% 68 

FAT 75% 133 

'\ 

6 I 

7 I 

9 t 

4 2 

0 2 

2 

2 2 

3 2 

4 2 

f 2 

6 2 

IT 

i : 

[ 5 

l l 

) A 

t t 

r t 


1 \ 

3 ‘ 

l l 

0 

DIET 




















77? 


72 



RATIO 

(00 

40 

%0 

70 

60 

SO 

40 

SO 

20 

JO 

0 



















/ 
























/ 
























r 





























































s* 







































































































































































total 

ACETONE 

0.5 

0.4 

0.3 

0.2 

0.1 

0 


































I 

i 























1 

V 























/ 

\ 

s 






















j— 


7? 

\ 
















A 























r“ 



















































Chart 3 . 

tions of urinary nitrogen made during this period there was more found 
than was contained in the food taken, but on the day when the difference 
was largest, Mar. 1, the ratio used to express the ketogenic balance had a 
value of 56 per cent when calculated from the food fed and of 59 per cent 
when the protein burned was calculated from the urinary nitrogen; this diff¬ 
erence is almost certainly within the limits of experimental error. 

This case showed the smallest excretion of the acetone bodies of any 
in the series (the scale which has been used in plotting the acetone excre¬ 
tion is ten times as great as it is in the other cases), but still, when diets 
approximating the 10, 10, 80 per cent, diet were fed, the concentrations of 
























































Case III 


386 


Studies on Acetonuria 





CO 

>0 

10 






to 


CO 


to 

rH 

CO 

O 


00 


• 

* 

rH 

CO 

CM 

0 

CO 

o> 

0 

05 

rH 

05 

rH 

CM 

CM 

rH 

05 


O 



8 

!>> 

0 

rH 

t-_ 

rH 

rH 

CO 

rH 

cO 

05 

to 

1 ^ 

rH 

05 

05 

cO 

CO 

rH 

00 



0 

d 

d 

rH 

rH 

rH 

rH 

rH 

d 

rH 

0 

rH 

O 

O 

0 

O 

rH 

co 


±; 2 
"O'C 

>>fc 

O 

O 

2.0 

10 

rH 

CM 

CO 

CO 

(M 

O 

to 

co 

CO 

05 

to 

rH 

rH 

CO 

to 

05 

0 

00 




CM 

rH 

O 

CO 

0 

to 

CO 

rH 


O 

co 

CO 

rH 

05 

O 

05 

0 


s 


rH 

Cl 

rH 

rH 

tH 

CM 

rH 

CM 

CM 

rH 

CM 

rH 


CM 

CM 

to 




CO 

CM 

CO 


00 

to 

0 

CO 

GO 

O 

00 

to 

05 

05 

00 

rH 

00 

to 



• 

Cl 

05 

CM 

rH 

0 

1- 

CO 

rH 

CM 

CM 

0 

05 

rH 

to 

CO 

GO 

to 


*1“ • 

8 

0 

O 

CM 

rH 

rH 

rH 

to 

0 

co 

CO 

CO 

to 

CO 

rH 

CO 

CM 

rH 

rH 


ca> 



• 
















O 

Jh 4 -> 

s ^ 


0 

d 

d 

O 

d 

d 

d 

0 

d 

d 

d 

0 

0 

O 

d 

O 

d 

rH 


0 




















0 0 

0 




















-g.S 

0 

Cl 

i> 

CO 

CM 

CO 


CM 


00 



01 

b- 

rH 

CO 

05 





0 

>-i 

CO 

CO 

rH 

CO 

05 

CO 

05 

CO 

rH 

GO 

d 

GO 

05 


co 

00 

05 

GO 




rH 

rH 

1> 

to 

rH 

CO 

0 

CO 

0 

05 

GO 

05 

to 

rH 

00 

O 

CO 



S' 





rH 


rH 


rH 







CM 

rH 

<D 


8 


















G 



0 

CO 

tO 

CO 

CO 

0 

CO 

to 

05 

GO 

05 

rH 

co 

i> 

CO 

CO 

rH 

CO 

Sh 

N»HN 

• 

T—1 

Cl 

04 

0 

rH 

0 

05 

0 

CO 

on 

rH 


CM 

rH' 

to 

CM 

1^ 



s 

CO 

tO 

rH 

10 

CO 



to 

rH 

CM 

CM 

CO 

CO 

to 

rH 

rH 

tO 

05 




© 

d 

d 

d 

d. 

d 

d 

0 

O 

O 

O 

d 

0 

d 

O 

O 

d 

O 




10 

0 

CO 

0 

CO 

CM 

CM 

CM 

rH 

05 

to 

0 

GO 

co 

CM 

CO 

CM 

00 


"N Pm 

s 

rH 

0 

CO 

rH 

rH 

rH 

i> 

rH 

CM 

CO 

rH 

CM 

CO 

co 

rH 

CO 


1^ 


• 














05 


CS 

00 

CO 

r- 

CO 

CO 

rH 

GO 

rH 

1> 

CO 

rH 


to 

cO 

to 

CO 

to 



>-* 

0 

0 

CM 

CO 

co 

05 

rH 

CO 

CM 

r- 

O 

05 

00 

CM 

00 

CO 

CO 

r- 


•pioy 


GO 

CO 

Cl 

O 

CO 

rH 

rH 

CM 

CM 

rH 

CO 

05 

rH 

00 

05 

tO 

r- 

0 


O 

O 

CM 

CM 

CO 

CO 

CM 

CM 

CM 

rH 

CM 

rH 

rH 

rH 

rH 

CM 

rH 

rH 

rH 

CO 


•UOT^O'BO'^ 

&3 

1> 

rH 

»o 

CO 

CO 

CO 

to 

to 

CO 

to 

GO 

tO 

O 

CO 

CO 

CO 

0 

to 

1> 

i>- 

i> 



a 

to 

tO 

CO 

to 

to 

to 

to 

to 

co 

CO 

CO 

CO 

CO 

to 

to 

to 

to 

to 




lO 

O 

0 

0 

O 

0 

to 

0 

to 

to 

0 

to 

0 

0 

0 

0 

0 

to 


•aumjOA 


tH 

CO 

r—( 

CO 

05 

CM 

05 

rH 

rH 

r- 

rH 

I- 

to 

0 

CO 

01 

CO 

GO 



GO 

10 

CO 

to 

CO 

rH 

OC 

O 

GO 

to 

CO 

co 

CO 

GO 


CO 

CO 

co 

Alveo¬ 

lar 

CO2. 

8 


rH 

rH 

CO 

rH 

rH 

to 

O 

rH 

rH 




CO 


0 

1^ 

rH 

s 


CO 

CO 

CO 

CO 

CO 

CO 

CO 

CO 

CO 

co 

co 

CO 

CO 


CO 

CM 

CO 

rC 


rH 

rH 


CM 

0 

O 

0 

CM 

O 


CM 

rH 

0 

rH 

0 

CM 


0 

# W) 


0 

0 


0 

d 

O 

0 

O 

O 


d 

d 

d 

d 

d 

d 


0 

£ 

rH 

rH 


rH 

rH 

rH 

rH 

rH 

rH 


rH 

rH 

rH 

rH 

rH 

rH 


rH 









rH 

rH 

rH 

05 

05 

05 

05 








*OOH B N 

8 






05 

05 

05 

00 

GO 

GO 

00 















rH 

rH 

rH 

CO 

CO 

CO 

CO 











CM 

Cl 

CM 

CM 

CM 

CM 

CM 

CM 

CM 

CM 

CM 

CM 

CM 

CM 

CO 

CO 

CO 





O 

0 

0 

O 

O 

O 

0 

O 

O 

O 

O 

O 

O 

O 

05 

05 

05 


•sst.ioj'bq 


C-* 

tO 

10 

to 

CO 

CO 

to 

to 

»o 

to 

tO 

to 

to 

to 

to 

rH 

rH 

rH 




H 

rH 

rH 

rH 

rH 

rH 

rH 

rH 

rH 

rH 

rH 

rH 

rH 

rH 

rH 

rH 

rH 


f 

V> *3 
§ § 

c* 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

to 

to 

to 



a c 


rH 

rH 

rH 

rH 

rH 

rH 

rH 

rH 

rH 

rH 

rH 

rH 

rH 

rH 

























-AqoqjBQ 























t- 

i^ 

w 




1^ 

1- 


1^ 





C 5 

05 

05 



8 

Cs 

c- 

CO 

CO 

CO 

CO 

CO 

co 

co 

co 

CO 

co 

co 

co 

CO 

co 

rH 

rH 

rH 






















Q 




0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

to 

to 

to 


‘Pjl 

«** 
Cn V 

c- 

00 

00 

00 

00 

00 

00 

00 

GO 

00 

00 

GO 

00 

00 

00 

00 

00 

00 


. 


rH 

rH 

rH 

rH 

rH 

rH 

rH 

rH 

rH 

rH 

rH 

rH 

rH 

rH 

rH 

rH 

rH 




8 

c* 

CO 

CO 

CO 

CO 

CO 

CO 

CO 

CO 

CO 

CO 

CO 

CO 

CO 

CO 

rH 

rH 

rH 



Cb 


rH 

rH 

rH 

rH 

rH 

rH 

rH 

rH 

rH 

rH 

rH 

rH 

rH 

rH 

rH 

rH 

rH 



. •« 
& s 

c« 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 


•uxa^ojj 

a 5 


rH 

rH 

rH 

rH 

rH 

rH 

rH 

rH 

rH 

rH 

rH 

rH 

rH 

t-H 

rH 

rH 

rH 









1- 

1^ 






r^. 








§ 

<2> 

c- 

CO 

CO 

CO 

CO 

CO 

co 

CO 

CO 

co 

CO 

CO 

CO 

co 

CO 

co 

CO 

co 















O- 



<>-• 

e-. 




rH 

CM 

CO 

rH 

to 

co 


00 

05 

0 

rH 

CM 

CO 

rH 

to 

CO 

r- 

00 


6 


r-H 

rH 

rH 

rH 

rH 

rH 

rH 

rH 

rH 

CM 

CM 

CM 

CM 

CM 

CM 

CM 

CM 

CM 



• 


















ce 

01 



















Q 


o 3 

>« 

w 

s 

V* 







N* 

X 




** 




s 




























































R. S. Hubbard and F. R. Wright 


387 


GO 

CO 

00 rb 

oo 

no 


00 

© 

© 


co 

© 

CO © 

CO 


b~ o 

C5 LO 

no 

r-H 


1- 

© 

© 

© 

© 


CO © 

CO 


© 

© o 

b* 03 

O 

t-H 

t-H 

t-H 

03 

© 

© 

03 

t-H 

© © 

© 


03 

r-i d 

d d 

o 

d 

d 

d 

© 

© 

r-H 

© 

© 

© © 

© 



O 

rh 

05 

no 

co 

© 

lb 



lb 

ib 

rb Ib 

Tb 


© 

b~ 1^ 

co co 

CO 

no 

co 

© 

© 

lb 

© 

© 


Tb b- 

© 


rH 

GO GO 

^ 03 


CO 

03 

co 

Tb 

t-H 

© 

CO 

rH 


CO 

t-H 

r-H 





03 

03 





rH 

no rb 

GO 00 

Tb 

00 

rH 

CO 

00 

w 

CO 

© 

CO 

© CO 

© 


»o 

03 b- 

rb 

oo 

co 

03 

Ol 

b- 

© 

b- 

© 

CO 

© © 



00 

O CO 

CO 03 

o 

o 

t-H 

rH 

rH 

Ol 

© 

t-H 

t-H 

© © 


© 

d d 

o d 

d 

o 

d 

© 

© 

© 

© 

© 

© 

© © 

© 



CO 03 

03 05 

o 

o 

00 

ib 

© 

rb 

03 

© 

03 

lb © 

© 


CO 

Tb 05 

rb no 

d 

t-H 

t-H 

© 

rH 

© 

CO 

03 

© 

© © 

00 


o 

lb Tf 

b- 03 

t-H 

03 

03 

03 

CO 

00 

© 

03 

rH 


r-H 
















o 

Tb 

b- 

00 

b- 

© 

oo 

© 

© 


03 

Tb © 

© 


03 

O CO 

1- r-H 

no 

uo 

Hb 

no 

t-H 

03 

03 

OO 

© 

00 00 

© 


C0 

1-H lb 

no no 

Tb 

CO 

CO 

CO 

© 

CO 

© 

CO 

03 

03 03 

03 


rH 

T—1 O 

o d 

d 

o 

o 

© 

© 

© 

© 

© 

© 

© © 

© 


CO 

GO ^b 

r-H O 

o 

03 

Tb 

1^ 

03 

© 

© 

© 

CO 

© 00 

03 


^b 

Tb Ttl 

00 i-H 

lb 

CO 

00 

© 

CO 

© 

03 

© 

CO 

© © 

rb 


ib 

lb no 

co co 

co 

Tb 

no 

© 

© 

Tb 

© 

© 

© 

© rb 

^b 


CO 

© »h 

03 1-t 

00 


Hb 

00 

© 

© 

CO 

Tb 

© 

N rH 

03 


05 

© CO 

05 CO 

CO 

05 

Hb 

CO 

Tb 

lb 

© 

Ol 

© 

O rH 

© 


03 

03 03 

t-H 03 

t-H 

t-H 

03 

03 

03 

rH 

CO 

CO 

03 

03 03 

rH 



© 



no 

TTb 


© 


© 

© 


© 



CO 

no b- 

05 05 

03 

GO 

00 

00 

00 

ib 

© 

© 

© 

03 rH 

03 


no 

no no 

no no 

CO 

no 

no 

© 

© 

© 

© 

© 

© 

© © 

© 


o 

O O 

o o 

O 

no 

no 

© 

© 

© 

© 

© 

© 

© © 

© 


03 

Tb CO 

t-H CO 

Hb 

Ol 

no 

© 

I'. 

03 

Ol 

© 

03 

Tb i-H 

03 


00 

GO lb 

no 05 

00 

CO 

no 

© 

© 

CO 

© 

00 

GO 

lb lb 

© 


GO 

O t-h 


03 

t-H 

05 

CO 

CO 

CO 

© 

© 

© 

© © 

© 


03 

CO CO 


CO 

CO 

Ol 

CO 

CO 

CO 

CO 

CO 

CO 

CO CO 

CO 
















© 















© 















rb 


03 

03 03 

03 no 

no 

no 

no 

© 

© 

© 

© 

rH 

rH 

t-H rH 

r-H 

t-H 

© 

o o 

O 05 

05 

05 

05 

© 

© 

© 

© 

© 

© 

© © 

© 

© 

t-O 

no no 

no xb 

rb 

^b 

Hb 

Tb 

© 

© 

© 

© 

© 

© © 

© 

© 



-H "H 













rH 

t-H t-H 

t-H t-H 

r-H 

t-H 

rH 

r-H 

rH 

t-H 

t-H 

t-H 

rH 

rH rH 

rH 

rH 








© 

© 

© 






o 

o o 

O no 

no 

no 

no 

© 

© 

© 

© 

© 

© 

o © 

© 

© 

t-H 

r-H r-H 

t-H t-H 

t-H 

t-H 

rH 

rH 




03 

03 

Ol 03 

03 

03 



b- CO 

cO 

co 

CO 

© 


b~ 

r- 

© 

© 

© © 

© 

to 

co 

co co 

CO no 

no 

no 

no 

© 

CO 

co 

co 

b- 

lb 

lb lb 

lb 

lb 

o 

o o 

O no 

no 

no 

no 

© 

rH 

t-H 

t-H 

© 

© 

o © 

© 

© 

GO 

00 GO 

oo b~ 

lb 

lb 

lb 


oo 

00 

oo 


b- 

lb lb 

lb 

Ib 

rb 

Tb -*b 

Tb no 

no 

no 

no 

© 

rH 

rH 

rH 

ib 

Ib 

b- Ib 

lb 

1^ 

CO 

CO CO 

CO 03 

03 

03 

03 

03 

Tb 

rb 

© 

t-H 

r-H 

rH t-H 

rH 

r-H 

r-H 

r-H r-H 

t-H r-H 

t-H 

t-H 

t-H 

r-H 

r-H 

r-H 

t-H 

r-H 

t-H 

rH rH 

rH 

t-H 








© 

© 

© 






o 

o o 

o © 

o 

o 

© 

© 

© 

© 

© 

© 

© 

© © 

© 

© 

t-H 

^H r-H 

t-H t-H 

^H 

r-H 

t-H 

t-H 




t-H 

rH 

r-H t-H 

t-H 

t-H 


i> b~ 

lb b- 



b- 

i- 


i- 

ib 

b- 

ib 

Ib b- 

ib 

ib 

CO 

co co 

co co 

co 

co 

co 

co 

CO 

CO 

co 

co 

co 

co co 

co 

co 









O- 




• 



05 

03 

30 

31 

r-H 03 

CO 

Tb 

no 

© 


00 

© 

10 

t-H 

t-H 

(M CO 

t-H t-H 

r-H 

to 

rH 

p<’ 

c3 

># S# 

P 

© S 

- 

- 


- 

- 

- 

- 

s# 

N* 

V* 

V# V 

** 


















o 


a 

o 

-h 

<x> 

o 


a 

H| 

o 


aj 

a 


o 


'G 

T3 

O 

cc 

co 

o> 

S—i 
© 
X 
a> 

o 

Sh 

c3 

to 

© 

o 

-Q 

o 

a 

o 

-H 

D 

O 

c3 

03 

©! 


o 

03 

a 

o 

• F-H 

•+3 

c3 

C 

a 

Pi 

03 

+3 

03 

<*-i 

O 

CO 

-l-> 

3 

03 

03 

PS 


































PR 10% 37 T*. jPRIO %37<J m ‘ fPR 10% 37 \PRlO%nr*' 

DIET A CH 10% 37 DIET B 03D • C H 5^ 13.6 OIETC K33‘ CH 15% SS'] DIET D E22- CH 10% "J A 

FAT »0%I34 FAT ? $"% /4( FAUSYollS FAT70%/H 

__ MARCH APRIL 


388 


Studies on Acetonuria 



Chart 4 


































































































































































































MARCH APRIL 

A -- 


R. S. Hubbard and F. R. Wright 389 



Chart 4A. 






















































































































390 


Studies on Acetonuria 


the acetone bodies varied from ten to one hundred times the values of 
those found when the subject was on a normal diet. It seems possible that 
the continued loss of weight and negative nitrogen balance noted in this 
case may be accompanied by a liberation of glycogen which would serve as 
an additional (endogenous) source of antiketogenic compounds, but as other 
experiments, as Case IV, do not show evidences of such a process during 
loss of weight this explanation can only be offered tentatively. The figures 
actually obtained show that the border-line diet was very close to the basal 
one, and as this is the only case in the series in which this was found to be 
true, it seems reasonable that some such phenomena as those suggested 
above may have diminished the excretion. The results show also that 
the ratio between the fat and carbohydrate rather than the amount of fat 
ingested determines the acetone excretion, for less acetone was found when 
the diet contained 153 gm. of fat and 45 gm. of carbohydrate than when it 
contained 133 gm. of fat and 36 gm. of carbohydrate. The excretion of the 
acetone bodies was so low that the determination of creatinine was not 
interfered with, and the determinations of this compound showed a high 
degree of success in collecting accurate 24 hour urines. 

Case III, Mrs. M. H., was a woman 28 years old, 5 ft. 3 in. tall, and weigh¬ 
ing 70 lbs. Her basal metabolism measured 1,240 calories per day. She 
had been suffering from severe chronic arthritis for 2 years, and was prac¬ 
tically helpless. A special nurse was assigned to the case, and both the 
nurse and the patient cooperated well in carrying out directions. Alto¬ 
gether the results of the study of this case were very satisfactory, but in 
some instances specimens of urine were unavoidably lost due to the condi¬ 
tion of the patient; the days on which these losses occurred are marked in the 
table with an interrogation point. The patient ate the entire amount of 
the diet provided at all times, and the diet furnished maintained the body 
weight throughout the experiment. It was possible to continue the base 
line diet long enough to determine the excretion of acetone caused by it 
with more accuracy in this case than in any other. Diets which had a lower 
ratio of carbohydrate to fat than did the basal diet caused a formation of 
larger amounts of acetone, and the change from one level of excretion to 
the other was gradual and not abrupt. 

A diet which had a ketogenic power of 108 per cent caused practically 
no increase in the excretion of acetone; one having a value of 78 per cent 
caused an excretion of distinctly increased amounts, although these 
amounts were not great; while the basal diet—-which has a value of 55 per 
cent—caused an excretion of between 1 and 2 gm. From these figures it 
would seem that the value of the border-line diet must lie at about 78 per 
cent, unless considerable importance is attached to the formation of very 
slight traces of the acetone bodies. 

In this case small amounts of sodium bicarbonate were fed over a period 
of a few days after acetone excretion was thought to have reached an 
equilibrium which corresponded to the basal (10, 10, 80 per cent) diet. 
The sodium bicarbonate lowered the excretion of ammonia and of titra- 


TABLE V 

Case V. 


) 

> 


R. S. Hubbard and F. R. Wright 391 





O 

GO 

rH 







to 

CO 

Tfl 

to 

CO 



rH 

rH 

© 

co 

co 

rH 





CM 

05 

CO 


CM 

CO 

05 

o 

o 

CO 

05 


to 




05 

t-H 


© 

© 

rH 



• d 

s 

O 

o 


CM 

rH 

rH 

CM 

CO 

oo 

oo 

CM 

o 

o 

o 



rH 

t-H 

t-H 

© 

© 

rH 



* 2 
o os 


o 

o 

d 

t-H 

t-H 

CM 

CM 

CM 

rH 

d 

O 

d 

o 

d 



o 

o 

© 

© 

© 

© 



^3 -r* 

o 

o 

© 

c© 

i> 








oo 

t-H 

00 

rH 



co 

rH 

rH 



rH 



<50. £ 


CM 

CM 

d 

co 

GO 

r- 

00 

rH 

o 

00 

t-H 

r-H 

i> 

C5 



GO 

00 

rH 

© 

© 

© 






t-H 

oo 

to 

1> 

o 

to 

r- 

t-H 

00 

to 



CM 



CO 

CM 

CO 

© 

© 

00 




§ 




t-H 

t-H 

CO 

rH 

CO 


r-H 







rH 

rH 





© 

t-H 

rH 

GO 

to 

rH 

to 

rH 

o 


00 

rH 

to 

O 



rH 

GO 



© 

rH 





8 


05 

00 

CO 

CM 

s 

to 

CO 

CO 

00 

CO 

CO 

o 



CO 

© 

rH 


rH 

© 



+ O 

s 

o 

CO 

CO 


05 

1> 

CO 


t-H 

o 

o 

t-H 



CM 

O 

rH 

CM 

CM 

CM 



c» 

© 

d 

o 

d 

d 

o 

d 

d 

d 

d 

d 

o 

d 

d 



O 

d 

© 

© 

© 

© 



<D ^ 


























a a> 

























6 

c 

• H 

Sh 

o 2 

-*_> 03 

o” 

o 

Q> 

00 

CM 

CM 

o 

o 




co 

co 

o 

GO 

o 

co 




© 

00 

© 

© 

CM 


t5 


>-* 

d 

05 

GO 

CO 

CM 

o 

05 

CM 

o 

CO 

d 

d 

to 

00 



CO 

to 

© 

rH 

© 

© 







rH 

CO 

05 

CM 

CM 

CM 

oo 

00 



to 

CO 



rH 

CM 

CM 

00 

© 

rH 




if 






rH 

t-H 

t-H 
















rH 

CO 

cO 

00 

GO 

co 

8 


oo 

o 

TP 

o 

00 

© 




OO 

© 

CO 

CO 




Z 


t-H 

o 

GO 

t-H 

00 

CO 


r- 

CO 

CM 

to 

GO 

(© 




© 

© 


© 




W 

s 

CO 

CO 

to 

to 

rH 


to 


TtH 

co 

CO 

t-H 

rH 

rH 




t-H 

CO 

CM 

CM 




z 

C2> 

o 

d 

d 

d 

d 

d 

d 


o 

d 

d 

o 

d 

d 




© 

© 

© 

© 




^“4 



© 

o 

o 

o 

o 

o 


co 

o 

o 

o 

o 

05 





© 

CM 





tz 

8 


o 

t-H 

CM 

l> 


o 


00 

o 

o 

to 

05 

rH 





© 

I> 





H 



© 

oo 

1> 

to 

d 

to 


co 

to 

co 

t-H 

CM 

rH 





rH 

CM 





Vol¬ 

ume. 


o 

o 

o 

O 

0 

o 

8 

o 

o 

o 

o 

o 

O 

o 



8 

© 

© 

© 

© 

© 




05 


CM 

CO 


i> 

CM 




00 

O 

CO 



© 


rH 

t-H 

CO 




© 


00 

00 

00 


to 

co 


to 

to 

r-H 


CM 



to 

CO 

© 

CO 

rH 

© 


i 

O 

N 

s 


CO 





CO 


CO 



00 



^H 



rH 



rH 




8 


rH 





CO 


CO 



CO 



CO 



CO 



rH 



■4* 



CO 


o 


CM 


rH 


to 




GO 



o 


co 

CM 



© 

.5? 

kg, 


h- 




d 


CO 


CO 




CO 





h- 

h~- 



1> 

£ 



© 


to 


to 


to 


to 



to 

to 



to 


© 

© 



© 





CO 

05 

o 

t-H 

05 

co 

CM 

00 

o 

to 

o 

o 

o 


CO 

o 

CM 


© 

8 

© 



i 

O oo 

'S 2 



t-H 

GO 

05 

o 


CO 

to 

05 


CM 

to 

to 

o 

05 

rH 

o 

CO 

© 

© 

© 




C"~ 

o 

05 

00 

t- 

oo 

o 

o 

05 

o 

o 

CM 

CM 

CO 

rH 


oo 

oo 

t- 

00 

oo 

© 

O' 


O u 



cm” 

r-H 

r-H 

t-H 

t-H 

cm” 

cm” 

t-H 

cm” 

”cT 

"cm" 


rH 

rH 

rH 

rH 

t-H 

rH 

rH 

t-H 

t-H 



6 

£ 


05 

t-H 

to 

to 

CO 

00 



to 


00 

00 


CO 

00 


© 

rH 

rH 

rH 

© 




u 

e- 









• 











O’ 


H 

f* 


00 

d 

05 

o 

d 

CO 

CO 


CO 

CO 

o 

d 

co 

t-H 


CO 

to 

© 

© 

© 

H- 



T3 



t-H 

t-H 

t-H 

rH 

rH 

CM 

CM 

CM 

CM 

CO 

CM 

CM 

CM 

CM 

OJ 

CM 

CM 


























.4 

O 


























rO 

t-t 

8 

CO 

o- 

© 

to 

to 

to 

to 

CM 

cm 

CM 

CM 


r- 






© 

00 

CO 

00 

fr* 

O’ 


c5 

o 


rH 

rH 

rH 

rH 

rH 

1> 




rH 

t-H 

r-H 

rH 

rH 

rH 

CO 

CO 

CO 

CO 

© 

© 

© 

© 

© 


O 


Is 

O’ 

CM 

00 



00 

CO 

to 


00 

t-H 

CM 

CM 


rH 

05 


CM 


© 

© 

CM 

O’ 



b 


CM 

r-H 

t-H 

05 

d 





C5 

rH 

rH 

rH 

d 

t-H 

CO 

rH 

CO 

CO 

CO 

CM 


Q 



GO 

oo 

GO 


00 

1> 

i> 

i> 


cO 


i>- 

CO 

to 

co 

CO 

© 

© 

© 

© 

© 




$« 






















o3 


























Ph 



rH 

rH 

o 

05 


rH 

CO 

o 

oo 

CO 

oo 

00 

O 

rH 

05 

© 

CM 

© 

© 

o 

00 




£ 

O’ 

00 

GO 

l- 

rH 

CO 


t- 

t- 


to 



<o 

CM 

rH 

to 

© 

rH 

© 

© 

CO 

CV- 



S: 


t-H 

t—H 

t-H 

t-H 

t-H 

t-H 

rH 

rH 

r-H 

rH 

rH 

rH 

rH 

rH 

rH 

rH 

rH 

rH 

rH 

rH 

rH 




-*o 

8 


05 

t-H 

to 

to 

CO 

05 

00 

o 


05 

o 

o 


co 

CO 

CO 

CM 

CM 



CM 




§ 

O’ 







• 


• 







• 



• 


00 

05 

d 

o 

d 

00 

00 

d 

00 

oo 

GO 

00 

o 

05 

o 

d 

© 

© 

© 

© 

© 



a 








rH 


rH 

rH 

rH 

rH 

t-H 

rH 

rH 



*3 

a 

























-4-> 

O 


























£ 

£? 

O’ 

© 

co 

to 

to 

to 

to 

to 

to 

to 

to 

to 

to 

to 

to 

to 

to 


© 

© 

© 

© 

O’ 


s2> 


rH 

rH 

rH 

rH 

rH 

rH 

rH 

Ttl 





rH 

CO 

TH 

rH 

rH 

rH 

rH 

rH 

rH 




© 

t-H 

t-H 

CM 

CO 

rH 

to 

CO 


00 

05 

O 

rH 

CM 

CO 

rH 

to 

© 


00 

© 

© 

rH 

O 


CO 

CO 




rH 

t-H 

rH 

rH 

rH 

rH 

rH 

rH 

rH 

rH 

CM 

CM 

-4-> 

























a 

Ci 

>, 


nr 





















Q 








v* 




V# 


V# 



V* 

V* 

V* 



V# 








s# 

>■* 


*•* 


V# 


V * 


V* 





s * 



** 

** 

** 



»”5 


< 






















Results of determinations on the acetone bodies are expressed in terms of acetone. 





































392 


Studies on Acetonuria 


table acid, diminished the degree of acidity of the urine, and increased the 
tension of carbon dioxide in the alveolar air. The amount of the acetone 
bodies and their concentration in the urine were increased while the subject 
received the drug, and returned to the level previously established after 
it was discontinued. The periods before and after the alkali was given 
were short, but the changes were so marked that the experiment probably 
indicates a real increase in the excretion of these compounds. The results 
are similar to those recorded by Joslin 4 and by Forssner (1911). As this 
was the only case in the series in which the effect of the administration of 


DIET A 



CA 1.1870 


CAL1022 


’CAL 2 I^O 


■1 

PR 1 

CM 10 %4S 

diet b mrm 

PR<f % 45^«. 

CH 14% J1 

DIET C 

PR 8 Vo 4b'4 m - 
CH 21 % 1/7 

01ET 0 


PAT 80% 16? 


PAT77 % 16? 


FAT no 



CAL 1740 
PR \0.4%4Si 
CH ir.fi 
PAT 7S' 


JULY_AL»ftOST 


30 51 I 2 M r i M 1 10 II It 0 M Ifli n I* M 10 



1 Ml! 


















DIET 


r~ 



m 

an 

m 

m 

mm 


zz 

as 

SS 


ss 

52 

52 


as 


3 


kw5 

140 

130 

no 

1 >0 

too 

40 

to 

70 

60 

50 

40 

30 

20 

10 

0 




















































I 







































































— 





t 

— 



















~] 



I 

7 

7 
















_ 


t 



j 

r~ 

7 

3 




' 

7* 













1 



S 




















7 





















Z\ 

s 

□ 










j 









—< 




J 




























: 























f - 























_ 


























































TOTAL f m ’ 

ACETONE 3 

2 

0 







i 


H 




















2 

s 

z 

S 


















y 






s; 


~ 


— 






1 






z 








53 

L 


! 


b 

~ 







□ 

□ 

—I 













— 

_ 



z 





Chart 5. 


alkali was studied, it is impossible to do more than note what may be an 
accidental finding. 

Case V, Miss M. G., was a woman 22 years old, 5 ft. 2\ in. in height, 
who weighed 127 lbs. Her basal metabolism measured 1,430 calories per 
day. This experiment was much less satisfactory than those described 
above; the patient failed to eat all of the food provided in any of the diets, 
and also, apparently, to collect 24 hour urines in a satisfactory manner. 


4 Joslin (1917), pp. 394 and 395. 











































































TABLE VI. 

Case VI. 


R. S. Hubbard and F. R. Wright 


393 


>> 

•fcs 

O c3 

PH 

T3 

■ 


3 


© r -1 £r ‘OCO^rjHCOCOOOrtiOcC'iOOO 

^2 N O >0 OCOOiOTj<QC'TtTt'^Ort<iO 

CO ’“H Ol Ol H^^CONlOlMCO^HHCO 

© © ooo ©©©ddddddddd 


o 

o 

o 

co 


N- 

i> 

»0 

r- 

CO 

rh 

<N 

CO 

CO 

rt< 


1> 

CO 


no 


rH 

d 

05 

05 

no 

co 

CO 

no 

N 


<N 

CO 


CO 

no 

rH 

cO 

cs 

s 

r-H 

r- 

rH 

rH 

<N 

rH 

N 

01 


<N 

CO 

N 


no 

rH 

rH 

Ol 


V 

o 

+ 

0> 

c 

o 

-u 

0> 

o 


w 

55 


<N t- 
<N 05 

lO r—I 

d d 


CO 

o 

Ol 


o 

no 

<N 


O rfH 
05 t-H __ 
h Ci IN CO 


MOOOrfiMOOlN 
O500WNONM00 
CO CO CO CO (N 

o o o o o 


o o o o o 


S3 

05 


o o 


o 

c> 


O <N 

<N <N 
Th <N 


CO 

CO 

CM 


O OOICCONOOOOOOIMOOCOMH 

>0 ONOOHIOOO^OHCO^ 

Ol HHHlNHNlOiOCO^HH 


H. 

CO 


o 

no 

CO 


CO 

t-H 

O 

o 


c3 , 

I* 


no 

d 



a> 


O 

o 


o 

no 

o 


3 

o 

o 

no O 

o o 

© 

© 

© 

© 

© 


s 

. 


05 


b- 

no 



Ol 

05 

8 8 

O CD 

00 © 

N 

Ol 

oo 


© 


j3 


<N 

00 


oo 

O 

© 


<N 

Ol 

no 


00 

© 

N 

CO 


O 

> 


rH 




rH 

rH* 


rH 

rH 

rH 

rH rH 

rH rH 




rH 

rH 

•H 

A 




Th 





no 






CO 





_hC 


i 







l>- 






h- 





o 

£ 



no 





no 






»o 








o 

O 

Ol 

Ol 

<N 

00 

00 

Tt* 

o 

o 

N O 

© Ol 

© 

© 


© 

© 


6 rA 


no 

no 

Ol 

Ol 

Ol 


CD 



nO 

00 no 

no t* 

no 

no 

no 


no 




IN 

Ol 

Ol 

<N 

Ol 

rH 

rH 

© 

rH 

Ol 

i-H <N 

Ol Ol 

IN 

Ol 

Ol 

N 

<N 

U * 


rH 

rH 

rH 

rH 

rH 

rH 

rH 

rH 

rH 

i-T 

rH rH 

rH rH 

rH 

rH 

i— r 

rH 

rH 


a? 

H> 

1 

<N 

<N 

05 

05 

05 

i> 

Ol 

rH 

rH 

<N 

Ol <N 

Ol CD 

Th 

rH 

CO 

rH 



c3 

(h 


05 

05 

00 

00 

00 

00 

05 

rH 

d 

05 

o os 

d oo 

01 

<N 

Ol 

oi 

<N 


TJ 

>> 

a 

rH 

rH 

rH 

rH 

rH 

rH 

rH 

N 

Ol 

rH 

Ol i—< 

rH rH 

Ol 

Ol 

Ol 

N 

<N 


I 

c3 

O 




















s 

ca 

09 

09 

58 

00 

no 

8 8 S 

55 

59 

09 

3 3 

60 

58 

70 

70 

70 

69 

70 


o 

5 




*• OOOO^^^NNiOH 


Tt< rtn CO CO CO no rJH 
cO CO CO CO CO CO CO 


oococooocoooooco 

' i 


CO rh no 
CO CO CO 


TtH lO 
CO CO 


CO CO 


00 

CO 


s OOOOOcOTfcOO?OcO 

£ 05 05 05 05 05 00 GO l>“ 00 05 GO 


o © o o o o o 

05 05 05 05 05 05 05 


.5 

*3 

o 

- 


N N N CO H ^ 00 01 h QO GO H 05 00 

£ COCOTti^TjitOOWCOCO^OCOCONNCON’d 

rH rH rH rH rH rH rH rH rH H H rH rH rH rH rH rH t—H rH 

A 


00n0i0n0c0t^»0050t^00000i— iOO 
iOnOTfi , ^TtiTti' , ^CO^t | nO' , cti»OnO*OTtirtirti' , ^'^ti 


a 

Q 


ClWrti0C0N 00 05O 
01<N<M<N<M<N<N<NC0 


OICO^iOCONOOOSOh 
r— It—Ii—I t—I »— I rH i— I i—<N<N 

P VPV\P\PNP'P'#'P'PV#'#'P'J'*'P'^V#'4 


Results of determinations of the acetone bodies are expressed in terms of acetone. 





































394 


Studies on Acetonuria 


Although the weight varied somewhat from day to day it was fairly well 
sustained throughout the period of study. It seems probable in such a case 
that fat not taken in the diet is replaced in metabolism by tissue fat, 
although the possibility of an increased combustion of glycogen must also 
be considered. This case showed acetonuria when the diet had a keto- 
genic balance of about 80 per cent, and the acetonuria practically cleared 
up when a diet having a balance of 110 per cent was fed. 



DIET A ZZA ■ 

CAL 120 0 

Ch 

'AT67.f%?8 

JULY 

|CAL»2.3-0 

Jpr 12.2% 40 9m. 

|CH 224% 70 
(FAT 64.3% 90 

1 

1 

3 

4 

S 1 

6 l 

7 

S 1 

1 2 

0 2 

,1 2 

1 2 

5 2 

4 2 

f 2 

6 2 

7 1 

1 2 

1 3 

<0 3 

1 

DIET 

Z 

77? 


7Z7. 

77 

22 

22 

22 


77/ 


7 Z 


77 


2S 






RATIO 

MO 

'30 

110 

110 

100 

10 

80 

70 

io 

90 

• 40 

30 

20 

10 

0 

















































































































































































































































































































































TOTAL 1*- 

ACETONE 2- 

1 

0 

































































Chart 6. 


Case VI, Mrs. E. Y., was a woman 70 years old, 5 ft. 2| in. tall, who 
weighed 135 lbs. Her basal metabolism measured 1,185 calories per day. 
There was a special nurse assigned to the case, but there was a lack of coop¬ 
eration because the patient had a prejudice against protein food, and 
disliked large quantities of fat. This case was even less satisfactory than 
the preceding one. When the series of experiments was commenced the 
patient had been living on a diet low in carbohydrate for some time as a 
part of treatment for chronic arthritis. Acetone was found in her urine 
by the qualitative test used (Legal’s) when the diets did not seem to be 
severe enough to cause the presence of the compound, and it seemed desira- 






















































R. S. Hubbard and F. R. Wright 


395 


ble to determine whether there was an increased elimination of both the 
acetone bodies. There is no doubt that the patient showed such an 
increase, although no strictly normal values are available for comparison. 
The diet taken during the period of study contained, on the average 15 
per cent of the calories as protein, 17 per cent as carbohydrate, and 68 per 
cent as fat, and had a ketogenic balance of 120 to 130 per cent. The results 
are different from those found on other subjects, and may perhaps be 
attributed to changes in the metabolism of the patient caused by her 
advanced age. 

There is one fact which is evident in all of the experiments; 
when the diet was changed the level of the acetone excretion 
changed to correspond, but changed gradually. Why these 
changes should have taken 3 or 4 days in some instances cannot 
be explained in an entirely satisfactory way. One factor which 
delayed the response was undoubtedly the time which it took in¬ 
gested fat to pass through the digestive, assimilative, and meta¬ 
bolic processes, but this did not seem adequate to account for 
the delay completely. It is possible that when there was a large 
excess of ketogenic compounds included in the diet, glycogen or 
other antiketogenic materials were furnished from the reserve 
supplies of the body in larger amounts than normal. If this was 
so not only would the changes be gradual, as was found to be the 
case, but also the amounts of acetone found during the period 
for which a given diet was fed would be lower than that expected 
from a calculation of the ketogenic balance of the diet. The data 
reported above are not sufficient to decide this question. What¬ 
ever may have been the cause of this gradual change in the ace¬ 
tone excretion, there are three facts which result from it: first, 
no decision concerning the acetone excretion which corresponds 
with a diet can be made until the diet has been fed for several 
days; second, analyses of the fat content of stools is not necessary 
because it would not be possible to decide to what acetone ex¬ 
cretion the figures would apply; third, it is useless to feed the 
diets—at least to feed the fat content of the diets—in small 
amounts taken frequently. 

In interpreting the meaning of the excretion of acetone when 
diets are fed, which are at or near the border-line of ketogenic 
antiketogenic equilibrium, there are certain possibilities which 
must be kept in mind. For instance, the body tissues may fur¬ 
nish part of the material burned, and this will be of ketogenic or 


396 


Studies on Acetonuria 


antiketogenic nature, as it may be derived from fat, protein, or 
glycogen. It is probable that a mixture of these is consumed in 
amounts which will appreciably affect the excretion of acetone 
when the subject is losing weight, and may so affect it at other 
times. The composition of the different materials burned may 
vary with the general nutrition of the patient, and with other 
factors not understood, factors which are perhaps similar to those 
which affect the storage of fat. 

Another fact which must be taken into consideration, especially 
when slight increases of acetone excretion are studied, is the 
probable variation in the mixtures of foodstuffs burned in the 
body at different times during the 24 hour periods. In the study 
of the effect of diets on the excretion of the acetone bodies dis¬ 
cussed here the interpretation has been based on the analysis 
of 24 hour specimens of urine; the results of these analyses have 
been discussed as if they represented not only the total, but also 
the average excretion for the periods. Such an assumption is 
not correct, because when the ketogenic and antiketogenic com¬ 
pounds are present in equivalent amounts, or when the antike¬ 
togenic material is present in excess, no matter how great that 
excess may be, no acetone bodies will be formed, while if the 
ketogenic material is in excess they will be formed. 

Although the nature of the results showed that it could not 
make much difference how the fat in any given diet was fed, it 
did seem possible, because carbohydrate is more rapidly assim¬ 
ilated, that feeding small amounts of this foodstuff frequently 
would reduce the error described above. In one of the cases in 
the series the carbohydrate was given at six times during the 
day, but practically the same amount of acetone was found as 
when it was given in three meals. 

It is possible that different combinations of foodstuffs may be 
simultaneously oxidized in different parts of the organism. If 
the cells were burning mixtures of foodstuffs which were at or 
near the border-line of ketogenic antiketogenic equilibrium, it is 
conceivable that slight disturbances of the blood and nutriment 
supplied to various parts of the body would lead to a local pro¬ 
duction of acetone. A similar explanation has been suggested 
to account for the rise in blood acetone found after the injection 
of small amounts of adrenalin chloride (Hubbard and Wright, 


R. S. Hubbard and F. R. Wright 


397 


1921). It does not seem probable that this effect can be of as 
much importance in producing an excretion of acetone as can 
differences in metabolism at different times during the day, but 
the possibility that there is some such source of acetone cannot 
be neglected. If the age of the patient studied in the sixth 
experiment has any effect upon the formation of the acetone 
bodies it was probably to exaggerate the effect either of the 
“local” or “temporary” production of those compounds. 

The various uncertain factors which must influence the inter¬ 
pretation of the results of these experiments may be summarized 
as affecting three different parts of the study: one, as influencing 
the value used to express the ketogenic balance of the diets; 
two, as rendering uncertain the accuracy with which the excre¬ 
tion of the acetone bodies observed corresponds to that which 
should have been found; and, three the interpretation of the 
results in terms of ketogenic equilibrium. Uncertainties which 
affect the value of the ratio include: one, the use of a figure 
to convert the sum of the antiketogenic factors into terms of 
fatty acid which is based on the molecular weights of the higher 
fatty acids; two, the use of the total carbohydrate content of the 
diet instead of its glucose equivalent in calculating the antiketo¬ 
genic compounds; three, the use of the fat fed instead of the fat 
absorbed for measuring the amount of the ketogenic compounds; 
and, four, the uncertainty as to the correct percentage of pro¬ 
tein which figures as a source of antiketogenic material. Of these 
uncertainties the first is the only one which would certainly 
increase the apparent ketogenic value of the expression, the sec¬ 
ond and third sources of error would certainly increase its antike¬ 
togenic value, while the effect of the fourth is undeterminable. 
The errors which may have affected the experiments themselves 
are of various kinds: first, the subjects did not always eat all of 
the food which was provided; second, they may have eaten arti¬ 
cles of food which were not provided; third, enough protein was 
not eaten in all cases exactly to maintain the subjects in nitrogen 
equilibrium; fourth, enough food was not taken in all instances to 
maintain metabolic equilibrium; fifth, 24 hour specimens of urine 
were not always accurately collected. The first three sources of 
error mentioned were such as would lead to real or apparent in¬ 
creases in the amount of antiketogenic compounds metabolized, 


THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. L, NO. 2 


398 


Studies on Acetonuria 


as fat was the food which was left untouched, carbohydrate was 
the food which the subjects most craved, and a negative nitrogen 
balance was observed more frequently than was a positive one; 
the effect of the loss in weight and of the failure to collect urines 
accurately cannot be determined. A detailed examination of 
these sources of error has shown that either they could not be 
wholly avoided, or that their influence on the results was slight. 

The difficulties which affect the interpretation of the excretion 
of the acetone bodies influence the study of the results of the 
experiments more than do the other uncertainties met with in these 
experiments. These sources of uncertainty include: temporary 
production of the acetone bodies due to variations in the food¬ 
stuffs burned at different times during the day; local production 
of the acetone bodies caused by variations in different parts of the 
organism; and, possibly, the effect of glycogen drawn from the re¬ 
serve stores of the body. All of these except the last would lead 
to a production of acetone greater than the composition of the 
diets would indicate. 

In interpreting the value of the expression 

jqq ^ 1.5 (weight carbohydrate + 25 per cent weight protein) _ jq- er cen ^. 

95 per cent weight fat ~ ** 

which will express the condition of ketogenic antiketogenic equili¬ 
brium, the effect of these uncertainties, particularly of those af¬ 
fecting the interpretation of small amounts of the acetone bodies, 
must be kept in mind. When the value of the expression was 
100 per cent or more, acetone was not found in the urine except id 
very small amounts, and in two of the cases studied, the excre¬ 
tion decreased when the diets had this value to the normal level. 
In one other case such a diet failed to cause the appearance of a 
distinctly increased acetonuria, although the period of study (3 days) 
was perhaps not long enough to produce an equilibrium in the 
body. When diets were fed which gave numerical values between 
55 and 60 per cent rather large amounts of acetone were excreted; 
there was a distinctly increased excretion also, except in Case II, 
when diets giving values of about 80 per cent were taken. It seems 
most reasonable to attribute the small amounts of acetone found 
on the diets which figured at 100 per cent to local and temporary 
production of the acetone bodies, and to conclude that values of 



R. S. Hubbard and F. R. Wright 


399 


80 to 90 per cent approximately represent the diet in which the 
ketogenic and antiketogenic foods are present in equivalent 
amounts. 

It has been shown already that certain numerical values of 
the expression when ketogenic equilibrium is attained correspond 
to the different possible antiketogenic effects of the glycerol radi¬ 
cle: if glycerol does not figure as a source of antiketogenic com¬ 
pounds the value is 100 per cent; if glycerol is converted into glu¬ 
cose, and this glucose takes part in the reaction between ketogenic 
and antiketogenic compounds, the value is 83 per cent; if glycerol 
takes part in the reaction as a three carbon atom residue, the 
value is 67 per cent. A comparison of these values with the one 
which has been found experimentally to correspond with the con¬ 
dition of equilibrium makes it seem most probable that the gly¬ 
cerol residue of the fats does figure only to the extent to which it 
can yield glucose. These conclusions would be expressed mathe¬ 
matically as follows: 

1.5 (weight carbohydrate + 25 per cent weight protein) 00 

100 X-^- 7 - • , , £ , - = 83 per cent 

95 per cent weight fat 

If this equation is transposed so as to express the amounts of pro¬ 
tein, fat, and carbohydrate which should be fed to produce a 
condition of ketogenic equilibrium, the expression becomes: 

1.9 (weight carbohydrate + 25 per cent weight protein) = fat. 

This expression is practically identical with that stated by Wood- 
yatt (1921) : 5 “2 X carbohydrate + protein — fat.” 

It is of course possible that too much stress has been laid upon 
11 temporary” and “local” sources of traces of acetone, and that 
not enough emphasis has been placed upon glycogen as a source 
of antiketogenic compounds. If very small amounts of acetone 
result from an excess of antiketogenic material in the diet, 100 
per cent probably represents the condition of equilibrium. In 
this case the expression would be: 

1.5 (weight carbohydrate + 25 per cent weight protein) _ 1QQ cent 
X 95 per cent weight fat 

and the expression for the relative amounts of food would become: 


Woodyatt (1921), p. 133. 




400 


Studies on Acetonuria 


1.42 (weight carbohydrate+25 per cent weight protein) = weight fat. 

This expression probably does not express the condition of keto- 
genic equilibrium correctly; the one given above is almost certainly 
preferable. 

It seems reasonable to conclude from the experiments reported 
that the two and three carbon atom residues from the a-amino- 
acids do not figure directly in the antiketogenic reaction, but are 
condensed to glucose. If these residues did react with the keto- 
genic compounds the numerical value for each diet would be higher 
than they are reckoned here; acetonuria would develop and clear 
up at values of from 100 to 120 per cent, and traces of acetone 
would be found in some cases when the value was 150 per cent. 

The charts and tables recording these experiments have been 
examined for evidence of an adaptation of the organism to these 
diets high in fat with a consequent reduction of the amounts of 
the acetone bodies excreted. Folin and Denis (1915) have re¬ 
ported evidences of such an adaptation to starvation in three obese 
women studied by them, but there did not seem to be such a re¬ 
sponse to diets high in fat. When the basal diet—containing 10 
per cent of the calories in the form of protein, 10 per cent in the 
form of carbohydrate, and the balance in the form of fat—was 
resumed after periods during which diets containing relatively more 
or less fat was fed, the excretion of acetone returned to the level 
first established if the base line diet was continued over a sufficient 
period. 

The method adopted of plotting the concentration of the acetone 
bodies upon paper ruled with logarithmic characteristics shows 
clearly the relationship between the two fractions of the acetone 
bodies discussed in an earlier paper (Hubbard, 1921). When 
large amounts of the acetone bodies were excreted the acetone 
from /3-hydroxy butyric acid was in excess of that from preformed 
acetone plus acetoacetic acid, but when the concentrations were 
only slightly increased the two fractions were as a rule nearly equal; 
in some cases the acetone from preformed acetone plus acetoace¬ 
tic acid was in excess. When acetonuria developed slowly it 
was this fraction which increased first, while the /3-hydroxybutyric 
acid increased later. The interpretation of these facts is com¬ 
plicated by differences in the kidney thresholds of the different 
acetone bodies. 


R. S. Hubbard and F. R. Wright. 


401 


Other results which have not yet been discussed include changes 
in alveolar carbon dioxide tension, the excretion of ammonia and 
of titratable acid, and changes in the reaction of the urine. The 
urinary ammonia roughly paralleled the acetone bodies except 
when sodium bicarbonate was added to the diet; during that per¬ 
iod the excretion of ammonia was markedly reduced, while that 
of the acetone bodies was somewhat increased. The alveolar 
carbon dioxide tension was somewhat lowered by the more extreme 
diets, and the values returned to normal when sodium bicarbon¬ 
ate was taken. The variations of the titratable acidity and hy¬ 
drogen ion concentration were little greater than those which 
are normally found; these were, of course, markedly affected by 
the administration of the alkali. 

CONCLUSION. 

A method has been suggested for expressing the ketogenic bal- 
lance of any diet mathematically. A series of six experiments 
has been described in which the effect of diets high in fat on the 
excretion of the acetone bodies by normal subjects was studied, 
and the results compared with this mathematical expression. 
From the results obtained the following conclusions have been 
drawn: (1) that the mechanism which controls the formation 
of increased amounts of the acetone bodies can be regarded as a 
molecular reaction or balance between ketogenic substances such 
as the fatty acids and antiketogenic substances such as glucose; 

(2) that protein figures as an antiketogenic compound only to 
the extent of the glucose which it can yield in the organism; 

(3) that glycerol, when fed as a part of the fat molecule figures 
as an antiketogenic compound only to the extent to which it 
forms glucose in the organism; and (4) probably that glycerol 
so fed does figure as an antiketogenic compound to the extent 
to which glycerol itself can yield glucose. 

Our thanks are due to Dr. Philip A. Shaffer for suggestions 
offered and for encouragement extended during the progress of 
the work described. 


402 


Studies on Acetonuria 


BIBLIOGRAPHY. 

Acree, S. F., Mellon, R. R., Avery, P. M., and Slagel> E. A., J. Infect. Dis., 
1921, xix, 7. 

Benedict, F. G., Boston Med. and Surg. J., 1918, clxxviii, 667. 

Folin, O,, Laboratory manual of biological chemistry, New York and 
London, 1916. 

Folin, O., and Bell, R. D., J. Biol. Chem., 1917, xxix, 329. 

Folin, O., and Denis, W., J. Biol. Chem., 1915, xxi, 183. 

Folin, O., and Denis, W., J. Biol. Chem., 1916, xxvi, 473. 

Folin, O., and Wu, H., J. Biol. Chem., 1919, xxxviii, 81. 

Forssner, G., Skand. Arch. Physiol., 1909, xxii, 349. 

Forssner, G., Skand. Arch. Physiol., 1911, xxv, 338. 

Fridericia, L. 8., Hospitalstid., 1914, lvii, 585. 

Hubbard, R. S., J. Biol. Chem., 1921, xlix, 357. 

Hubbard, R. S., and Wright, F. R., J. Biol. Chem., 1921, xlix, 385. 

Joslin, E. P., The treatment of diabetes mellitus, Philadelphia and 
New York, 2nd edition, 1917. 

Joslin, E. P., Diabetic manual, Philadelphia and New York, 2nd edition, 
1919. 

Lusk, G., The elements of the science of nutrition, Philadelphia and 
London, 3rd edition, 1917. 

Marriott, W. McK., J. Am. Med. Assn., 1916, lxvi, 1594. 

Morris, J. L., J. Biol. Chem., 1915, xxi, 201. 

Pemberton, R., Am. J. Med. Sc., 1917, cliii, 678. 

Pemberton, R., and Foster, G. L., Arch. Int. Med., 1920, xxv, 243. 

Poulton, E. P., Brit. Med. J., 1915, ii, 392. 

Shaffer, P. A., J. Biol. Chem., 1921, a, xlvii, 433. 

Shaffer, P. A., J. Biol. Chem., 1921, b, xlvii, 449. 

Woodyatt, R. T., Arch. Int. Med., 1921, xxviii, 125. 























* 



































*• 











































































